Methods and compositions for treating neurodegenerative disorders and alzheimer&#39;s disease and improving normal memory

ABSTRACT

The disclosure relates generally to neurodegenerative disorders and more specifically to a group of presenilin/G-protein/c-src binding polypeptides and methods of use for modulating signaling and progression of Alzheimer&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/399,516, filed Feb. 17, 2012 (now U.S. Pat. No. 8,951,794), which isa continuation of U.S. patent application Ser. No. 12/464,850, filed May12, 2009 (now U.S. Pat. No. 8,129,334), which is a continuation-in-partand claims priority to U.S. patent application Ser. No. 12/264,872,filed Nov. 4, 2008 (abandoned), which application is acontinuation-in-part of U.S. application Ser. No. 11/693,926, filed Mar.30, 2007 (now U.S. Pat. No. 7,851,228), which application claimspriority to U.S. Provisional Application Ser. No. 60/788,524 filed Mar.31, 2006, the disclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH

This invention was made with Government support under Grant Nos.AG017888, NS055161, NS27580, and NS44768 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE DISCLOSURE

The invention relates generally to treating neurodegenerative disordersand more specifically to a group of presenilin/G-protein/c-src bindingpolypeptides and small molecule drugs designed to modulate thephysiologic interactions of polypeptides required for the production ofβ-amyloid (Aβ).

BACKGROUND

Alzheimer's disease (AD) is a degenerative disorder of the human centralnervous system characterized by progressive memory impairment andcognitive and intellectual decline during mid to late adult life. Thedisease is accompanied by a variety of neuropathologic featuresprincipal among which are the presence in the brain of amyloid plaquesand the neurofibrillary degeneration of neurons. The etiology of thisdisease is complex, although in about 10% of AD cases it appears to befamilial, being inherited as an autosomal dominant trait. Among theseinherited forms of AD, there are at least four different genes, some ofwhose mutants confer inherited susceptibility to this disease. The σ4(Cys112Arg) allelic polymorphism of the Apolipoprotein E (ApoE) gene hasbeen associated with AD in a significant proportion of cases with onsetlate in life. A very small proportion of familial cases with onsetbefore age 65 years have been associated with mutations in the β-amyloidprecursor protein (APP) gene on chromosome 21. A third locus associatedwith a larger proportion of cases with early onset AD has recently beenmapped to chromosome 14q24.3. The majority (70-80%) of heritable,early-onset AD maps to chromosome 14 and appears to result from one ofmore than 20 different amino-acid substitutions within the proteinpresenilin-1 (PS1). A similar, although less common, AD-risk locus onchromosome 1 encodes a protein, presenilin-2 (PS-2, highly homologous toPS-1). Based upon mRNA detection, the presenilins appear to beubiquitously expressed proteins, suggesting that they are normallyhousekeeping proteins required by many cell types.

Presenilin 1 is a 43-45 kDa polypeptide and presenilin 2 is a 53-55 kDapolypeptide. Presenilins are integral proteins of membranes present inhigh molecular weight complexes that are detergent sensitive. Threeprotein components of the complexes in addition to presenilin are known.

SUMMARY

The disclosure provides methods and compositions for identifying agentsthat modulate activity of presenilins. Accordingly, the methods andcompositions provided herein may be used to modulate the production ofAβ in the brain by (1): interfering with the binding of theextra-cellular N-terminal domain of β-APP with PS-1 or PS-2; or (2) byusing as an inhibiting agent a small peptidomimetic molecule, or a smallfragment of an antibody molecule directed to an epitope on either theinteracting surfaces of the β-APP or PS molecules. In one aspect, thepeptide is a soluble N-terminal domain of PS-1 or -2.

In one embodiment, a method of identifying an agent that modulatespresenilin G-protein coupled receptor (GPCR) activity is provided. Themethod includes a) contacting presenilin, or fragment thereof, with aG-protein under conditions that would permit binding of the G-protein topresenilin; b) prior to, simultaneously with, or subsequent to a),contacting presenilin, or fragment thereof, with an agent; c) monitoringpresenilin-mediated binding to the G-protein; and d) determining whetherthe agent modulates presenilin binding to the G-protein therebyidentifying an agent that modulates presenilin G-protein coupledreceptor (GPCR) activity. In some aspects the modulating is byinhibition of presenilin binding to the G-protein. In other aspects, themodulating is by activating presenilin binding to the G-protein. Thepresenilin can be presenilin-1 (PS-1) or presenilin-2 (PS-2). TheG-protein can be G_(o), G_(s), G_(i), G_(z) or G_(q).

The disclosure further provides methods of treating Alzheimer's Diseaseor inhibiting the onset of Alzheimer's Disease comprising contacting asubject with an agent identified by the methods described above.

In some aspects, the agent includes a naturally occurring or syntheticpolypeptide or oligopeptide, a peptidomimetic, a small organic molecule,a polysaccharide, a lipid, a fatty acid, a polynucleotide, an RNAi orsiRNA, an asRNA, or an oligonucleotide.

The methods provided herein may be conducted in vitro or in vivo. Insome aspects, a method further includes contacting the presenilin withβ-APP prior to, simultaneously with, or subsequent to contacting thepresenilin with the G-protein.

In another embodiment, a method of identifying an agent that modulatespresenilin-mediated Src protein kinase activity is provided. The methodincludes a) contacting presenilin, or fragment thereof, with β-APP underconditions that would permit binding of β-APP to presenilin; b) priorto, simultaneously with, or subsequent to a), contacting presenilin, orfragment thereof, with an agent; c) monitoring presenilin-mediated Srcprotein kinase activity; and d) determining whether the agent modulatespresenilin-mediated Src protein kinase activity.

Also provided herein are compositions and methods for treatingneurodegenerative disorders, and more specifically to a group ofpresenilin/G-protein/c-src binding polypeptides and small molecule drugsdesigned to modulate the physiologic interactions of polypeptidesrequired for the production of β-amyloid (Aβ), the oligopeptide that isthe primary neurotoxic agent in Alzheimer's disease (AD). The disclosureprovides methods and compositions that reduce the amount of Aβ in thebrain to an extent that significantly decreases the neurotoxicity in AD,or delays the onset, or decreases the severity of the disease. Suchmethods and compositions are useful for modulating signaling andprogression of Alzheimer's Disease and improve memory.

The disclosure also provides a method of inhibiting the production of Aβwith a small molecule agent that inhibits the interaction of PS-1 orPS-2 with the G-proteins G_(oA) and G_(oB). The cytoplasmic C-terminaland other domains of PS-1 or PS-2 have been shown to be the sites ofinteraction of G_(oA) and/or G_(oB) with PS, and that this G_(o)-PSintracellular binding is required for subsequent Aβ production,presumably via the downstream results of this binding process.

The disclosure similarly provides a method of inhibiting the productionof Aβ by contacting a cell expressing a PS-1 and/or PS-2 with an agentthat interferes with the downstream results of PS-1 and/or PS-2 bindingto G_(o) such as G_(o) activation with phospholipase C.

The disclosure also provides a method of inhibiting the production of Aβby the use of small molecules, peptides or antibodies selected tointerfere with the activities of members of the Src family of tyrosinekinases.

The disclosure also provides a method of inhibiting the production of Aβby the use of small molecules, peptides or antibodies selected tointerfere with the interaction between a PS-1 and/or PS-2 and a β-APP.

The disclosure further provides a method of assaying for inhibitors ofAβ production in a cell culture system consisting of a first transfectedcell type expressing β-APP but no PS mixed with a second cell typeexpressing PS but no β-APP. The inhibitory effect of an agent added tothis mixed cell culture would be measured from the activities of severallikely downstream effects of (a) the G_(oA) and G_(oB) interaction withPS-1 and PS-2; or (b) the Src family of tyrosine kinases; or (c) theinteraction of N-terminal domain of βAPP with the N-terminal domain ofPS-1 and/or PS-2.

The disclosure provides an isolated polypeptide consisting essentiallyof the amino acid sequence of an N-terminal fragment of a Presenilin-1or -2 polypeptide. In one embodiment, the isolated polypeptide consistsessentially of an amino acid sequence selected from the group consistingof: (i) N-DEEEDEEL-COOH (SEQ ID NO:5), (ii) SEQ ID NO:5 furtherincluding 1-50 additional amino acids at either the N- or C-terminal endwherein the peptide inhibits cell-cell interaction, inhibits Aβproduction or binds to a β-APP, (iii) N-RRSLGHPEPLSNGRP-COOH (SEQ IDNO:6), (iv) SEQ ID NO:6 further including 1-5 conservative amino acidsubstitutions, wherein the peptide inhibits cell-cell interaction,inhibits Aβ production or binds to a β-APP, (v) a sequence of (iii) or(iv) further including 1-50 additional amino acids at the N- orC-terminus, wherein the peptide inhibits cell-cell interaction, inhibitsAβ production or binds to a β-APP, (vi)N-RRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAK-COOH (SEQ ID NO:7), (vii) SEQID NO:7 further including 1-5 conservative amino acid substitutions,wherein the peptide inhibits cell-cell interaction, inhibits Aβproduction or binds to a β-APP, (viii) a sequence consisting of (vi) or(vii) further including 1-50 additional amino acids at the N- orC-terminus, wherein the peptide inhibits cell-cell interaction, inhibitsAβ production or binds to a β-APP, and (ix) any of the foregoingcomprising an unnatural amino acid or D-amino acid wherein the peptideinhibits cell-cell interaction, inhibits Aβ production or binds to aβ-APP. In one embodiment, the peptide comprise an amino acid sequence ofthe N-terminal fragment of about 5-80 amino acids in length and having asequence as set forth in SEQ ID NO:2 or 4 from amino acid 1 to aboutamino acid 80. In another embodiment, the peptide may be linked to asecond peptide useful for purification, or formation of oligomers.

In another embodiment the disclosure provides an oligomer comprising atleast two peptide that interact with a presenilin or β-APP. The at leasttwo peptides may be the same or different or may be directlyfused/linked or fused/linked by a linking domain or peptide.

The disclosure also provides an isolated polynucleotide consistingessentially of a nucleotide sequence encoding a peptide or oligomerdescribed herein. The polynucleotide can be incorporated in to anexpression vector. The polynucleotide or expression vector may betransfected or transformed into a host cell.

The disclosure provides a method for expressing a polypeptide asdescribed above and herein, comprising culturing a recombinant host cellinto which a polynucleotide encoding the polypeptide has beenintroduced.

The disclosure also provides a method of inhibiting the production of Aβcomprising contacting a cell with an interfering agent that interfereswith the intercellular binding of β-APP and presenilin-1 (PS-1) and/orpresenilin-2 (PS-2) or the activation of a G-protein. In one embodiment,the interfering agent comprises an extracellular domain of apresenilin-1 or -2. In another embodiment, the extracellular domaincomprises an N-terminal region of PS-1 or PS-2, or oligomers thereof. Inyet another embodiment, the interfering agent comprises a solubleN-terminal domain of PS-1 or -2. In yet another embodiment, theinterfering agent comprises an amino acid sequence selected from thegroup consisting of: (i) N-DEEEDEEL-COOH (SEQ ID NO:5), (ii) SEQ ID NO:5further including 1-50 additional amino acids at either the N- orC-terminal end wherein the peptide inhibits cell-cell interaction,inhibits Aβ production or binds to a β-APP, (iii) N-RRSLGHPEPLSNGRP-COOH(SEQ ID NO:6), (iv) SEQ ID NO:6 further including 1-5 conservative aminoacid substitutions, wherein the peptide inhibits cell-cell interaction,inhibits Aβ production or binds to a β-APP, (v) a sequence of (iii) or(iv) further including 1-50 additional amino acids at the N- orC-terminus, wherein the peptide inhibits cell-cell interaction, inhibitsAβ production or binds to a β-APP, (vi)N-RRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAK-COOH (SEQ ID NO:7), (vii) SEQID NO:7 further including 1-5 conservative amino acid substitutions,wherein the peptide inhibits cell-cell interaction, inhibits Aβproduction or binds to a β-APP, (viii) a sequence consisting of (vi) or(vii) further including 1-50 additional amino acids at the N- orC-terminus, wherein the peptide inhibits cell-cell interaction, inhibitsAβ production or binds to a β-APP, and (ix) any of the foregoingcomprising an unnatural amino acid or D-amino acid wherein the peptideinhibits cell-cell interaction, inhibits Aβ production or binds to aβ-APP. The disclosure also provides a method of inhibiting theproduction of Aβ by contacting a cell expressing PS-1 and/or PS-2 withan agent that inhibits the interaction of PS-1 and/or PS-2 with aG-protein. In one embodiment, the agent interacts with the C-terminaltail and/or other cytoplasmic domain of PS-1 and/or 2 with a G_(oA)and/or G_(oB).

In another aspect, the disclosure provides a method of improvingcognitive function and/or memory in a subject. The method includesadministering an agent that inhibits the interaction of PS-1 and/or PS-2with G-protein, G_(oA) and G_(oB). In one approach, the agent interactswith the C-terminal tail and/or other cytoplasmic domains of PS-1 and/or2 that interact with G_(oA) and/or G_(oB). The agent may also interferewith the downstream results of PS-1 and/or PS-2 binding to G_(o) such asG_(o) activation with phospholipase C. In another approach, the agentinhibits the activity of members of the Src family of tyrosine kinasesin cells expressing PS-1 and/or PS-2. In each case the agent would beadministered in an amount to improve cognitive function and/or memoryretention compared to a control subject.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a representative study to determine if PS-1 is a GPCR.Extracts of different cell cultures were analyzed in order to determinewhether G_(o) interacts with PS-1, including the necessary controls. Ineach lane, the particular cell extracts were first immunoprecipitatedwith a monoclonal Ab (MAb) directed to PS-1; the immunoprecipitate wasthen dissolved and subjected to SDS-PAGE electrophoresis, and theresulting gel was Western blotted with an antibody directed to G_(o)(this antibody recognizes both G_(oA) and G_(oB)) Lane 1 is a control ofan extract of untransfected ES (PS-1^(−/−)/PS-2^(−/−)) cells. Asexpected, this extract showed that no G_(oA) (or G_(oB)) wasimmunoprecipitated with Ab to PS-1. Lane 2 is an extract of ES cells,that had first been transfected with PS-1 only, but not with G_(oA). Noprotein band was observed for G_(oA); this was another controlexperiment. Lane 3 is an extract of the ES cells transfected with bothPS-1 and G_(oA). In this extract, G_(oA) is immunoprecipitated alongwith the PS-1, showing that PS-1 was bound to G_(oA), but not G_(oB). IfPS-1 without its C-terminal “tail” (lane 4), which protrudes from themembrane into the aqueous intracellular compartment), is transfectedinto ES double null cells along with G_(oA) (lane 6), little or noG_(oA) is immunoprecipitated along with the PS-1 tailess, showing thatthe C-terminal domain of PS-1 is the principal region of G_(oA) bindingto PS-1.

FIG. 2 shows a Western blot of a similar experiment to that of FIG. 1but with PS-2 instead of PS-1. Lanes 2 and 4 show that tail-less PS-2,unlike tail-less PS-1, still binds G_(oA) (and G_(oB)), and thereforethat the binding sites for G_(oA) and G_(oB) are not confined to theC-terminal domain of PS-2, as is the case for PS-1 (FIG. 1) Lane 1 isuntransfected ES (PS-1^(−/−)/PS-2^(−/−)). Lane 2 is PS-2+G_(oA). Lane 3is Tail-less PS-2+G_(oA). Lane 4 is PS-2+G_(oB). Lane 5 is Tail-lessPS-2+G_(oB).

FIG. 3 involves an independent way of demonstrating G_(oA) binding toPS-1. [³⁵S]-GTPγS, an analog of GTP, makes a covalent bond to the activesite of a G-protein that is blocked by a prior reaction with Pertussistoxin (PTx). In column 2, there is shown an 8-fold increase in³⁵S-incorporation into G_(oA) that is immunoprecipitated with antibodyto PS-1, but not into G_(oB) (lane 4). Therefore, PS-1 binds to G_(oA)(that has reacted with [³⁵S]-GTPγS to identify it as a G-protein (column2), but also to a lesser extent to G_(oB) than to G_(oA) (column 4). The³⁵S bindings to G_(oA) and G_(oB) are blocked by prior treatment withPTx (column 3 and 5).

FIG. 4 is a graph depicting ³⁵SGTPγS incorporation in extracts of EScells transfected with cDNA for PS-2 and G-protein G_(oA).

FIG. 5 shows a plurality of panels of immunofluorescence microscopiclabeling of fixed cells. Row a) Double immunofluorescence microscopiclabeling of untransfected, fixed but not permeabilized, DAMI cells withprimary rat Mab #1563 to human PS-1 N-terminal domain (Panel 1) and FITCconjugated anti-rat IgG secondary antibody shows cell-surfaceimmunolabeling of endogenous PS-1 amino terminal domain. Panel 2 showsthe same cells do not express appreciable amounts of cell-surface β-APPwhen labeled with Mab #348 to the β-APP extracellular domain andTRITC-conjugated anti-mouse IgG secondary antibody (red). Panel 3 showsthe Nomarski images of cells in panels 1 and 2. Row b) DoubleImmunofluorescence microscopic labeling of β-APP-transfected, fixed butnot permeabilized, DAMI cells shows cell-surface expressed β-APP whenlabeled with Mab #348 to the β-APP extracellular domain andTRITC-conjugated secondary antibody (Panel 2). Panels 1 and 3, the samecells treated as for FIG. 5 row a. Row c) Immunofluorescence microscopiclabeling of PS-1-transfected, fixed but not permeabilized, DAMI cellsshows high expression of cell-surface PS-1 (Panel 1) but not β-APP(Panel 2) when labeled with the same primary and secondary antibodiesdescribed in a. Panel 3 shows the Nomarski image of cells in panels 1and 2. These experiments show that transfection of the DAMI cells withPS-1 does not call forth cell surface expression of β-APP. Row d)Immunofluorescence microscopic labeling of β-APP-transfected, fixed butnot permeabilized ES cells, double-null for PS-1 and PS-2. Cells showcell-surface expressed β-APP when labeled with Mab #348 to the β-APPextracellular domain and TRITC-conjugated secondary antibody (red; Panel2). Panel 1 shows the result of labeling with primary rat Mab #1563 tohuman PS-1 N-terminal domain and FITC conjugated appropriate secondaryantibody, indicating the expected absence of PS-1 on the surfaces of ESdouble-null cells. Panel 3 shows Nomarski image of cells in Panels 1 and2. Row e) Immunofluorescence microscopic labeling of untransfected,fixed but not permeabilized ES cells, double-null for PS-1 and PS-2.Cells show cell-surface expressed endogenous mouse β-APP when labeledwith Mab #348 to the β-APP extracellular domain and TRITC-conjugatedsecondary antibody (Panel 2). Panels 1 and 3 labeled as in d; no cellsurface labeling for PS-1 (Panel 1) is observed in these untransfectedES cells. Bar, 20 μm.

FIG. 6 shows that within minutes after mixing β-APP-only expressingtransfected ES cells with PS-1 only expressing transfected DAMI cells, atransient protein tyrosine phosphorylation process arises in the mixedcell culture, as detected by ELISA analyses of the cell extracts. Thisactivity peaked at ˜8-10 mins after mixing line (a). The same experimentcarried out in the presence of 25 μg purified soluble β-APP line (b) or25 μg purified peptide of N-terminal domain of PS-1 fused to FLAG line(c) showed none of the increases observed in line (a). The addition of25 μg of purified peptide of the non-specific N-terminal domain of PS-2fused to FLAG line (d), however, resulted in very similar transientincreases in protein tyrosine kinase activity to line (a).

FIG. 7A-D shows experiments to determine the nature of the tyrosinephosphorylating enzyme activity in FIG. 6. Src family kinase assay withsynthetic peptides. a and b: β-APP:PS-1 interaction with separatelytransfected DAMI cells as a function of time after mixing. Src kinaseactivity was assayed using the Src family substrate peptide{lys19}cdc2(6-20)-NH₂ (black bars) and control peptides{lys19Phe15}cdc2(6-20)NH2 (white bars) and{lys19ser14val12}cdc2(6-20)NH2 (gray bars) for both the β-APP:PS-1 (a)and control pcDNA3:PS-1 (b) interactions. c and d: β-APP:PS-2interaction with separately transfected DAMI cells as a function of timeafter mixing. Src kinase activity was assayed using the Src familysubstrate peptide {lys19}cdc2(6-20)-NH₂ (black bars) and controlpeptides {lys19Phe15}cdc2(6-20)NH2 (white bars) and{lys19ser14val12}cdc2(6-20)NH₂ (gray bars) for both the β-APP:PS-2 (c)and control pcDNA3:PS-2 (d) interactions.

FIG. 8A-B shows inhibition of tyrosine kinase activity. ELISAs todemonstrate tyrosine kinase activity of DAMI cells which had beenseparately transfected with β-APP and PS-1 and mixed in the presence andabsence of 10 μg/ml Herbimycin A (a) and 10 nM PP2 (b), as a function oftime after mixing.

FIG. 9A-B shows β-APP:PS-1 intercellular interaction: C-Src activity inextracts of mixed cells. a. Western Immunoblot. β-APP:PS-1 interactionswith mixtures of separately transfected DAMI cells. Western immunoblotwith primary anti-PTyr polyclonal antibodies (Panel 1) andanti-pp60c-src monoclonal antibodies (Panel 2) from the same experimentin which β-APP-transfected DAMI cells were mixed with PS-1-transfectedDAMI cells for 0-12 mins. Panel 3: Antibody labeling of controlpp60c-src protein with the pp60c-src antibodies. Panel 4: Westernimmunoblots with primary anti-PTyr antibodies, as in Panel 1, fromexperiments in which β-APP-transfected ES double-null cells wereinteracted with PS-1-transfected DAMI cells. b. Autoradiograph ofin-vitro phosphorylated proteins. Extracts of separately transfectedβ-APP and PS-1 DAMI cell mixtures at 0-12 mins after mixing were firstimmunoprecipitated with antibodies to c-Src and then phosphorylated invitro with γ³²P-ATP. Autophosphorylation reactions were subjected toSDS-PAGE followed by autoradiography.

FIG. 10A-B shows β-APP:PS-2 intercellular interaction: C-Src activity inextracts of mixed cells. a. Western Immunoblot. β-APP:PS-2 interactionin extracts of separately transfected and mixed DAMI cells as a functionof time after mixing. Panels 1 and 2: Same as FIG. 9a except thatPS-2-transfected DAMI cells replaced PS-1-transfected cells in theintercellular interaction with β-APP and cells were mixed from 1-20mins. b. Autoradiograph of in-vitro phosphorylated proteins. Sameextracts as in part a. Same as 5b except that PS-2-transfected DAMIcells replaced PS-1-transfected DAMI cells in the intercellularinteraction with β-APP.

FIG. 11A-D shows β-APP:PS-2 intercellular interaction: Activity of Lynand Fyn in extracts of mixed cells. a and b. Western Immunoblots:β-APP:PS-2 interaction. Western immunoblot with primary anti-Lynpolyclonal antibodies (a, Panel 1) and anti-Fyn polyclonal antibodies(b, Panel 1) from the same experiment in which β-APP-transfected DAMIcells were mixed with PS-2-transfected DAMI cells for 0-20 mins andextracts made. No change with time in concentration of either Lyn or Fynprotein was observed. Panel 2: Antibody labeling of control Lyn (a) andFyn (b) protein with their respective antibodies. c and d.Autoradiograph of in-vitro phosphorylated proteins: β-APP:PS-2interaction. Extracts of mixtures of β-APP and PS-2 mixed transfectedcells at 0-20 mins after mixing were first immunoprecipitated withantibodies to Lyn (c) or Fyn (d) and then phosphorylated in vitro with³²P-ATP. Autophosphorylation reaction products were subjected toSDS-PAGE followed by autoradiography.

FIG. 12 shows the ³⁵S-GTPγS incorporation in extracts of mouse brainthat could be immunoprecipitated with monoclonal antibodies to PS-1.

FIG. 13 shows the ³⁵S-GTPγS incorporation in extracts of mouse brainthat could be immunoprecipitated with monoclonal antibodies to PS-2.

FIG. 14 illustrates intracellular domains of PS.

FIG. 15 shows the effect of intercellular β-APP:PS interactions on Aβproduction.

FIG. 16A-C shows G-Protein activation results from specific β-APP:PSmediated cell-cell interaction for the production of Aβ. (A) ³⁵S-GTPgSincorporation to demonstrate activation of intracellular G-protein inhigh- and low-density β-APP:PS-1 co-cultures. ³⁵S-GTPgS incorporation inco-cultures of β-APP-only with PS-1-only cells at high density (lane 1),after immunoprecipitation with antibody K-20 directed to Ga_(o). Lane 1shows over 200% increase in ³⁵S-GTPgS incorporation above basal levels.(see below). Lane 2: The same cells when cultured at lower densitiesshowing 33% increase. Lane 3: The high density co-cultures in thepresence of PTx decreased ³⁵S-GTPgS binding to 38% below basal. Lane 4:High density co-culture in which β-APP was replaced by vector pcDNA3.Lane 5: High density co-culture in which PS-1 was replaced by pcDNA3.Both show low levels of G-protein activation, presumably due to thepresence of endogenous mouse β-APP or PS-1 present in the pcDNAtransfected cell. Data are presented as activation promoted byβ-APP:PS-1 intercellular interaction, expressed as a percentage, where0% is the ³⁵S-GTPgS binding in control, prepared by mixing extracts inequal amounts from untransfected ES null cells and untransfectedAPP^(−/−) fibroblasts. (B) and (C) Aβ Production in PS-1:13-APPco-cultures at different cell densities and in the presence or absenceof PTx. Co-cultures of β-APP-only with PS-1-only cells in the presenceof ³⁵S-met at the final numbers of 1.5×10⁷ (lanes 1 and 4), 0.3×10⁷(lanes 2) and 0.15×10⁷ (lanes 3). PTx (500 ng/ml) was added to the highdensity culture in lanes 4. (B) Equivalent amounts (100 μg protein) ofeach cell extract were immunoprecipitated with MAb 6E10, electrophoresedon bicene-tris gels and autoradiographed. The amount of Aβ produced percell decreased with decreasing cell density (B), lane 1 versus lanes 2and 3). The presence of PTx in the high density culture (B, lane 4)completely inhibited the Aβ produced in a similar culture not containingPTx (B, lane 1). (C) The densities of the cell cultures werephotographed.

FIG. 17A-B shows specific inhibition of β-APP:PS mediated cell-cellinteraction inhibits both G-Protein Ga_(o) activation and Aβ production.(A) ELISAs to measure Aβ production in the presence of PS-1 Peptide1-80. Co-cultures of β-APP-only and PS-1-only expressing cells carriedout in the presence and absence of increasing amounts of Peptide 1-80(0-3 μM). Aβ production was determined by ELISA and was inhibited byPeptide 1-80 in a dose-dependent manner. (B) GTPgS assays to measureG-protein activation in the presence of PS-1 Peptide 1-80. Co-culturesof β-APP-only and PS-1-only expressing cells carried out as in (A) abovein the presence and absence of increasing amounts of Peptide 1-80 (0-3μM). ³⁵S-GTPgS assays were carried out on extracts as a measure ofG-protein activation which was similarly inhibited by Peptide 1-80 in adose-dependent manner. Data are presented as inhibition of G-proteinactivation, expressed as a percentage, where 100% is the ³⁵S-GTPgSbinding in the β-APP:PS-1 co-cultures in the absence of Peptide 1-80.

FIG. 18A-C shows G-protein activation by the soluble ectodomain of β-APP(β-APPs). (A) Addition of partially purified β-APPs, at theconcentrations of β-APPs shown, to PS-1-only APP^(−/−) fibroblastcultures increased ³⁵S-GTPgS incorporation in a dose-dependent manner(curve 1); Similar concentrations of β-APPs added to cultures ofuntransfected APP^(−/−) fibroblasts expressing only endogenous levels ofPS resulted, as expected, in only modest increases in ³⁵S-GTPgSincorporation (curve 3); Addition of equivalent β-APPs-depletedpreparations (after pre-treatment with β-APP antibodies) yielded noincreases in ³⁵S-GTPgS incorporation when added to PS-1-transfectedAPP^(−/−) fibroblasts (curve 4). Addition of equivalent preparations ofβ-APPs similarly treated with irrelevant antibody (Goat anti-rat IgG)showed slightly lower values than the untreated sample (curve 2).Similar concentrations of β-APPs when added to untransfectedES(PS^(−/−)) cultures showed no significant increases in ³⁵S-GTPgSincorporation (curve 5). Data are presented as stimulation promoted byβ-APPs, expressed as a percentage, where 0% is the ³⁵S-GTPgS binding inthe absence of β-APPs. (B) SDS PAGE and Western blot of partiallypurified β-APPs. Lane 1 shows a Coomassie stained gel of the partiallypurified β-APPs (arrow) used in this work. Lane 2 is a Western blot ofthe same preparation with MAb 348 showing the β-APPs band. (C) Westernblot to show depletion of β-APPs in sample treated with MAb 348. Lane 1:untreated sample; Lane 2, MAb-treated sample, used in FIG. 18A.

FIG. 19A-B shows activation of Endogenous G-proteins in Rat Membranes.(A) Increasing concentrations (0-400 pM) of β-APPs added to solublizedrat hippocampal membranes (100 μg) for 15 min increased ³⁵S-GTPgSincorporation in a dose-dependent manner. (B) Similar addition of β-APPsto PS-depleted rat membranes which were first pre-treated with a mixtureof polyclonal Ab to PS-1 and PS-2. Addition of β-APPs to the PS-depletedmembranes showed no increase in ³⁵S-GTPgS incorporation. Data arepresented as stimulation promoted by β-APPs, expressed as a percentage,where 100% is the ³⁵S-GTPgS binding in the absence of β-APPs.

FIG. 20A-C shows direct coupling of human G-protein G_(o) to human PS-1cytoplasmic domain. (A) Extracts of variously transfected ES cells werefirst immunoprecipitated with anti-PS-1 loop MAb, andtheimmunoprecipitates were electrophoresed and Western blotted withG_(o) antibody K20 (recognizes G_(oA) and G_(oB).) Lane 1: UntransfectedES null cells; Lanes 2-6, ES null cells transfected with cDNA for: PS-1only (lane 2); both PS-1 and G_(oA) (lane 3); PS-1 and G_(oB) (lane 4).(B) 7-TM PS-1 showing location of cytoplasmic loop domains 1, 2 and 3and cytoplasmic C-tail. (C) Effect of PS-1 cytoplasmic peptide fragmentson ³⁵S-GTPgS binding to rat brain G_(o)-protein. Peptides (200 μM) wereincluded in the incubation mixture without pre-treatment, andaccumulation of the ³⁵S-GTPgS was determined. Data are presented asactivation promoted by the peptide expressed as a percentage, where 100%is the ³⁵S-GTPgS binding in the absence of peptide. Lane 1, no addedpeptide; lane 2, loop 1 residues 1-16, see Table 1 for a list ofpeptides); lane 3, loop 1 residues 17-32; lane 4, loop 2; lane 5, loop3; lane 6, residues 1-20 of the C-tail; lane 7, residues 21-39 of theC-tail. Only cytoplasmic loop 3 (lane 5) and peptide C(1-20) comprisingthe first 20 amino acids of the C-tail (lane 6) promoted a significant200% increase in activation of ³⁵S-GTPgS binding over samples withoutpeptide.

FIG. 21 shows heparin affinity chromatography of polypeptide isolated inthe methods of the disclosure.

FIG. 22, shows gel filtration chromatography for peptide and polypeptideseparation.

FIG. 23 shows the effect of G-protein minigene inhibitors on ³⁵S-GTPγSincorporation.

FIG. 24 shows the effect of G-Protein minigene inhibitors on Aβproduction.

FIG. 25A-C shows ³⁵S-GTPγS incorporation in the presence of variousintracellular domains for different G-Proteins. (a) Gα_(s) (b) G_(q),and (c) G_(s).

FIG. 26 shows inhibition of Aβ production following β-APP:PS-1intercellular interaction in the presence of synthetic peptides.Addition of 0-3 μM of only Peptides 4, 7 and 8 at the time of theco-culture of the β-APP-only ad PS-1-only cells inhibits cell-cellinteraction and Aβ production, as measured by ELISA assays. PEPTIDE 4:(15-mer) RRSLGHPEPLSNGRP (SEQ ID NO:5); PEPTIDE 5: (15-mer)SNGRPQGNSRQVVEQ (SEQ ID NO:15); PEPTIDE 6: (15-mer) RQVVEQDEEEDEELT (SEQID NO:16); PEPTIDE 7: (15-mer) DEEEDEELTLKYGAK (SEQ ID NO:17); PEPTIDE8: (8-mer) DEEEDEEL (SEQ ID NO:5).

DETAILED DESCRIPTION

As used herein, the singular forms “a,” “and,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a protein” includes a plurality of such proteinsand reference to “the cell” includes reference to one or more cellsknown to those skilled in the art, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The disclosure provides methods and compositions useful for treatingAlzheimer's Disease and disorders as well as neuronal development andactivity modulated by the interaction of a Presenilin with β-APP. Thedisclosure demonstrates that interaction of Presenilin-1 and/or -2 withβ-APP results in the generation of A. Furthermore that Presenilinsactivate G-proteins upon binding β-APP.

The disclosure demonstrates that the β-amyloid precursor protein, β-APP,and PS-1 or -2 are components of an intercellular signaling system. Oneor more forms of β-APP can specifically bind either to PS-1, or PS-2,via their extracellular domains that protrude from their respective cellmembranes. This binding in vivo induces an intercellular signaling eventof significance to normal neural physiology or development. A by-productof this transcellular molecular binding, processes of vesicle formation,cellular internalization, and proteolytic degradation are set in motionthat result in the formation and cellular release of Aβ and its slowaccumulation in regions of the brain.

The disclosure demonstrates that PS-1, PS-2 and β-APP play a role inintracellular signaling. These three proteins have been examined fortheir respective roles in the proteolytic fragmentation of β-APP to Aβthat involves the PS proteins either directly or indirectly. Inaddition, one or more forms of β-APP on one cell surface and PS-1 (orPS-2) on another may be specific ligand and receptor components of anintercellular signaling system with a role in normal physiology. Thedisclosure provides evidence that intercellular surface binding of β-APPto the PS proteins functions in normal physiology to induce a signalingprocess within one, or both, of the adherent cells, leading ultimatelyto a developmental outcome significant for the organism.

The term “amyloid beta peptide” means amyloid beta peptides processedfrom the amyloid beta precursor protein (APP). The most common peptidesinclude amyloid beta peptides 1-40, 1-42, 11-40 and 11-42. Other lessprevalent amyloid beta peptide species are described as x-42, whereby xranges from 2-10 and 12-17, and 1-y whereby y ranges from 24-39 and 41.For descriptive and technical purposes, “x” has a value of 2 to 17, and“y” has a value of 24 to 41.

The presenilins have a number of domains that can be identified by oneor more different predictive algorithms or by experimental proteolyticlysis experiments, solubility assays, and the like. Presenilin-1 (PS-1)has a number of domains as set forth below. It will be recognized thatthe domains may vary from 1 to 5 amino acids at either end dependingupon the organism that expresses the polypeptide. In one embodiment, aPS-1 N-terminal domain comprises residues x1 to about x2 of SEQ ID NO:2,wherein x1 comprises amino acid 1, 2, 3, 4 or 5 and x2 comprises aminoacid 79, 80, 81, 82 or 83 of SEQ ID NO:2. In one embodiment, anN-terminal domain fragment comprises a peptide of between 5 and 81 aminoacids in length (e.g., 5, 10, 20, 30, 40, 50, 60, 70 or 80 amino acidsin length). Such peptide fragments (e.g., soluble fragments) are usefulas βAPP binding agents or for the development of antibodies specific toan N-terminal extracellular domain of presenilin-1. The N-terminaldomain may further comprise all or a fragment of the first transmembranedomain (TM-1) comprising amino acids 82-100. In one embodiment, the TM-1domain comprises amino acid x2 to about amino acid x3 of SEQ ID NO:2,wherein x2 comprises amino acid 79, 80, 81, 82, or 83 of SEQ ID NO:2 andx3 comprises amino acid 98, 99, 100, 101 or 102 of SEQ ID NO:2. AnN-terminal fragment of presenilin 1 further comprising a fragment of thefirst TM domain of presenilin-1 can be used to produce a membrane boundcompetitive inhibitor, which lacks an active G-protein domain.

The first cytoplasmic loop 1 of presenilin-1 comprises amino acid x3 toabout x4 of SEQ ID NO:2, wherein x3 comprises amino acid 98, 99, 100,101, or 102 of SEQ ID NO:2 and x4 comprises amino acid 130, 131, 132,133 or 134. The second transmembrane loop of presenilin-1 (TM-2)comprises amino acids x4 to about x5 of SEQ ID NO:2, wherein x4comprises amino acid 130, 131, 132, 133 or 134 of SEQ ID NO:2 and x5comprises amino acid 152, 153, 154, 155 or 156 of SEQ ID NO:2. Thepresenilin-1 polypeptide further comprises a second extracellular domain(exoplasmic 1) comprises amino acids x5 to about x6 of SEQ ID NO:2,wherein x5 comprises amino acid 152, 153, 154, 155 or 156 of SEQ ID NO:2and x6 comprises amino acid 161, 162, 163, 164, or 165 of SEQ ID NO:2.Presenilin-1 further includes a third transmembrane domain (TM-3)comprising amino acids x6 to about x7 of SEQ ID NO:2, wherein x6comprises amino acid 161, 162, 163, 164 or 165 of SEQ ID NO:2 and x7comprises amino acid 182, 183, 184, 185, or 186. Presenilin-1 comprisesa second cytoplasmic loop (loop 3/cytoplasmic loop 2) comprising aminoacids x7 to about x8 of SEQ ID NO:2, wherein x7 comprises amino acid182, 183, 184, 185 or 186 of SEQ ID NO:2 and x8 comprises amino acid192, 193, 194, 195 or 196 of SEQ ID NO:2. A fourth transmembrane domain(TM-4) of presenilin-1 can be generally described as comprising aminoacids x8 to about x9 of SEQ ID NO:2, wherein x8 comprises amino acid192, 193, 194, 195 or 196 of SEQ ID NO:2 and x9 comprises amino acid211, 212, 213, 214 or 215 of SEQ ID NO:2. Presenilin-1 includes a thirdextracellular domain (Loop4/exoplasmic loop 2) comprising from about x9to about x10 of SEQ ID NO:2, wherein x9 comprises amino acid 211, 212,213, 214 or 215 of SEQ ID NO:2 and x10 comprises amino acid 217, 218,219, 220 or 221 of SEQ ID NO:2. A fifth transmembrane domain (TM-5) ofpresenilin-1 can be generally described as comprising amino acids x10 toabout x11 of SEQ ID NO:2, wherein x10 comprises amino acid 217, 218,219, 220 or 221 of SEQ ID NO:2 and x11 comprises amino acid 236, 237,238, 239 or 240 of SEQ ID NO:2. Presenilin-1 further includes a thirdcytoplasmic domain (loop5/cytoplasmic 3) comprising amino acids x11 tox12 of SEQ ID NO:2, wherein x11 comprises amino acid 236, 237, 238, 239or 240 of SEQ ID NO:2 and x12 comprises amino acid 241, 242, 243, 244 or245 of SEQ ID NO:2. A sixth transmembrane domain (TM-6) is generallyreferred to by the sequence comprising x12 to about x13 of SEQ ID NO:2,wherein x12 comprises amino acid 241, 242, 243, 244 or 245 of SEQ IDNO:2 and x13 comprises amino acid 260, 261, 262, 263 or 264 of SEQ IDNO:2. Presenilin-1 further includes a third cytoplasmic domain (loop6/exoplasmic 3) comprising a sequence x13 to about x14 of SEQ ID NO:2,wherein x13 comprises amino acid 260, 261, 262, 263 or 264 of SEQ IDNO:2 and x14 comprises amino acid 405, 406, 407, 408 or 409 of SEQ IDNO:2. Presenilin-1 further includes a seventh transmembrane domain(TM-7) comprising a sequence x14 to about x15 of SEQ ID NO:2, whereinx14 comprises amino acid 405, 406, 407, 408 or 409 of SEQ ID NO:2 andx15 comprises amino acid 427, 428, 429, 430 or 431 of SEQ ID NO:2. TheC-terminal cytoplasmic tail (C-tail/cytoplasmic) includes a sequence x15to about x16 of SEQ ID NO:2, wherein x15 comprises amino acid 427, 428,429, 430 or 431 of SEQ ID NO:2 and x16 comprises amino acid 462, 463,464, 465, 466 or 467 of SEQ ID NO:2.

PS-1 Residue Domain Numbers* Sequence NH2  1-81MTELPAPLSYFQNAQMSEDNHLSNTVR SQNDNRERQEHNDRESLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAKH TM-1  82-100 VIMLFVPVTLCMVVVVATI Loop 1101-132 KSVSFYTRKDGQLIYTPF (Cyto- TEDTETVGQRALHS plasmic 1) TM-2 133-154ILNAAIMISVIVVMTILLVVLY Loop 2 155-163 KYRCYKVIH (Exo- plasmic 1) TM-3164-184 AWLIISSLLLLFFFSFIYLGE Loop 3 185-194 VFKTYNVAVD (Cyto-plasmic 2) TM-4 195-213 YITVALLIWNFGVVGMISI Loop 4 214-219 HWKGPL (Exo-plasmic 2) TM-5 220-238 RLQQAYLIMISALMALVFI Loop 5 239-243 KYLPE (Cyto-plasmic 3) TM-6 244-262 WTAWLILAVISVYDLVAVL Loop 6 263-407CPKGPLRMLVETAQERNETLFPALIYSS (Exo- TMVWLVNMAEGDPEAQRRVSKNSKYN plasmic 3)AESTERESQDTVAENDDGGFSEEWEAQR DSHLGPHRSTPESRAAVQELSSSILAGEDPEERGVKLGLGDFIFYSVLVGKASATASGDWNTT TM-7 408-429 IACFVAILIGLCLTLLLLAIFC-Tail 430-467 KKALPALPISITFGLVFYFATDYLVQPFMDQ (Cyto- plasmic) *Refer toSEQ ID NO: 2

Presenilin-2 (PS-2) has a number of domains as set forth below. It willbe recognized that the domains may vary from 1 to 5 amino acids ateither end depending upon the organism that expresses the polypeptide.In one embodiment, a PS-2 N-terminal domain comprises residues x1 toabout x2 of SEQ ID NO:4, wherein x1 comprises amino acid 1, 2, 3, 4 or 5and x2 comprises amino acid 85, 86, 87, 88 or 90 of SEQ ID NO:4. In oneembodiment, an N-terminal domain fragment comprises a peptide of between5 and 87 amino acids in length (e.g., 5, 10, 20, 30, 40, 50, 60, 70 or80 amino acids in length). Such peptide fragments (e.g., solublefragments) are useful as βAPP binding agents or for the development ofantibodies specific to an N-terminal extracellular domain ofpresenilin-2. The N-terminal domain may further comprise all or afragment of the first transmembrane domain (TM-1) comprising amino acids87-106. In one embodiment, the TM-1 domain comprises amino acid x2 toabout amino acid x3 of SEQ ID NO:4, wherein x2 comprises amino acid 85,86, 87, 88 or 90 of SEQ ID NO:4 and x3 comprises amino acid 104, 105,106, 107 or 108 of SEQ ID NO:4. An N-terminal fragment of presenilin 1further comprising a fragment of the first TM domain of presenilin-2 canbe used to produce a membrane bound competitive inhibitor, which lacksan active G-protein domain.

The first cytoplasmic loop 1 of presenilin-2 comprises amino acid x3 toabout x4 of SEQ ID NO:4, wherein x3 comprises amino acid 104, 105, 106,107 or 108 of SEQ ID NO:4 and x4 comprises amino acid 136, 137, 138, 139or 140 of SEQ ID NO:4. The second transmembrane loop of presenilin-2(TM-2) comprises amino acids x4 to about x5 of SEQ ID NO:4, wherein x4comprises amino acid 136, 137, 138, 139 or 140 of SEQ ID NO:4 and x5comprises amino acid 158, 159, 160, 161 or 162 of SEQ ID NO:4. Thepresenilin-2 polypeptide further comprises a second extracellular domain(exoplasmic 1) comprises amino acids x5 to about x6 of SEQ ID NO:4,wherein x5 comprises amino acid 158, 159, 160, 161 or 162 of SEQ ID NO:4and x6 comprises amino acid 167, 168, 169, 170 or 171 of SEQ ID NO:4.Presenilin-2 further includes a third transmembrane domain (TM-3)comprising amino acids x6 to about x7 of SEQ ID NO:4, wherein x6comprises amino acid 167, 168, 169, 170 or 171 of SEQ ID NO:4 and x7comprises amino acid 187, 188, 189, 190 or 191 of SEQ ID NO:4.Presenilin-2 comprises a second cytoplasmic loop (loop 3/cytoplasmicloop 2) comprising amino acids x7 to about x8 of SEQ ID NO:4, wherein x7comprises amino acid 187, 188, 189, 190 or 191 of SEQ ID NO:4 and x8comprises amino acid 198, 199, 200, 201 or 202 of SEQ ID NO:4. A fourthtransmembrane domain (TM-4) of presenilin-2 can be generally describedas comprising amino acids x8 to about x9 of SEQ ID NO:4, wherein x8comprises amino acid 198, 199, 200, 201 or 202 of SEQ ID NO:4 and x9comprises amino acid 217, 218, 219, 220 or 221 of SEQ ID NO:4.Presenilin-2 includes a third extracellular domain (Loop4/exoplasmicloop 2) comprising from about x9 to about x10 of SEQ ID NO:4, wherein x9comprises amino acid 217, 218, 219, 220 or 221 of SEQ ID NO:4 and x10comprises amino acid 223, 224, 225, 226 or 227 of SEQ ID NO:4. A fifthtransmembrane domain (TM-5) of presenilin-2 can be generally describedas comprising amino acids x10 to about x11 of SEQ ID NO:4, wherein x10comprises amino acid 223, 224, 225, 226 or 227 of SEQ ID NO:4 and x11comprises amino acid 242, 243, 244, 245 or 246 of SEQ ID NO:4.Presenilin-2 further includes a third cytoplasmic domain(loop5/cytoplasmic 3) comprising amino acids x11 to x12 of SEQ ID NO:4,wherein x11 comprises amino acid 242, 243, 244, 245 or 246 of SEQ IDNO:4 and x12 comprises amino acid 247, 248, 249, 250 or 251 of SEQ IDNO:4. A sixth transmembrane domain (TM-6) is generally referred to bythe sequence comprising x12 to about x13 of SEQ ID NO:4, wherein x12comprises amino acid 247, 248, 249, 250 or 251 of SEQ ID NO:4 and x13comprises amino acid 266, 267, 268, 269 or 270 of SEQ ID NO:4.Presenilin-2 further includes a third cytoplasmic domain (loop6/exoplasmic 3) comprising a sequence x13 to about x14 of SEQ ID NO:4,wherein x13 comprises amino acid 266, 267, 268, 269 or 270 of SEQ IDNO:4 and x14 comprises amino acid 385, 386, 387, 388 or 389 of SEQ IDNO:4. Presenilin-2 further includes a seventh transmembrane domain(TM-7) comprising a sequence x14 to about x15 of SEQ ID NO:4, whereinx14 comprises amino acid 385, 386, 387, 388 or 389 of SEQ ID NO:4 andx15 comprises amino acid 407, 408, 409, 410 or 411 of SEQ ID NO:4. TheC-terminal cytoplasmic tail (C-tail/cytoplasmic) includes a sequence x15to about x16 of SEQ ID NO:4, wherein x15 comprises amino acid 407, 408,409, 410 or 411 of SEQ ID NO:4 and x16 comprises amino acid 443, 444,445, 446, 447 or 448 of SEQ ID NO:4.

PS-2 Residue Domain Numbers* Sequence NH2  1-87MLTFMASDSEEEVCDERTSLMSAESPTPRS CQEGRQGPEDGENTAQWRSQENEEDGEEDPDRYVCSGVPGRPPGLEEELTLKYGAKH TM-1  88-106 VIMLFVPVTLCMIVVVATI Loop 1107-138 KSVRFYTEKNGQLIYTTFTEDTPSVGQRLLNS (Cyto- plasmic 1) TM-2 139-160VLNTLIMISVIVVMTIFLVVLY Loop 2 161-169 KYRCYKFIH (Exo- plasmic 1) TM-3170-189 GWLIMSSLMLLFLFTYIYLG Loop 3 190-200 EVLKTYNVAMD (Cyto-plasmic 2) TM-4 201-219 YPTLLLTVWNFGAVGMVCI Loop 4 220-225 HWKGPL (Exo-plasmic 2) TM-5 226-244 VLQQAYLIMISALMALVFI Loop 5 245-249 KYLPE (Cyto-plasmic 3) TM-6 250-268 WSAWVILGAISVYDLVAVL Loop 6 269-387CPKGPLRMLVETAQERNEPIF (Exo- PALIYSSAMVWTVGMAKLDPSSQGALQLPYDPE plasmic 3)MEEDSYDSFGEPSYPEVFEPPLTGYPGEELEEEEE RGVKLGLGDFIFYSVLVGKAAATGSGDWNT TM-7388-409 TLACFVAILIGLCLTLLLLAVF C-Tail 410-448 KKALPALPISITFGLIFYFSTDNL(Cyto- VRPFMDTLASHQLYI plasmic) *Refer to SEQ ID NO: 4

The skilled artisan will recognize that the boundaries of these domainare approximate and that the precise boundaries of such domains, as forexample the boundaries of the transmembrane domains, may differ in 1-5amino acids from those predicted herein.

The most C-terminal residues of the cytoplasmic tail domains (along withother cytoplasmic domains) of Presenilin polypeptides are believed to beinvolved with interaction with G-proteins, such that substitutions ofthose residues are likely be associated with an altered G-proteinactivation or binding function, or with a lack of that function, for thepolypeptide.

As used herein, “β-APP binding polypeptide or peptide” includes humanPresenilin-1 (SEQ ID NO:2), variants (e.g., Presenilin-2; SEQ ID NO:4)and species homologues such as murine Presenilin-1 and fragments ofthese Presenilin polypeptides and their species homologues. Presenilinpolypeptides of the disclosure have biological activities and functionsthat are consistent with those of the other Presenilin familypolypeptides. Polypeptides of the Presenilin family are expressed incell types including neuronal cells throughout development. Typicalbiological activities or functions associated with this family ofpolypeptides are β-APP binding, G-protein activation and Aβ peptideformation. β-APP binding activity is found on the N-terminal domain andthe extracellular loops of the Presenilin polypeptide. G-proteinactivation is associated with the C-terminal cytoplasmic tail domain andother cytoplasmic domains of Presenilin polypeptides.

Presenilin polypeptides such as human Presenilin-1 have heterotypicbinding activity; each of these binding activities is associated withthe extracellular loop domains of Presenilin polypeptides. Thus, foruses requiring heterotypic binding Presenilin polypeptides of thedisclosure will include those having at least one extracellular loopdomain and exhibiting at least one such binding activity. Presenilinpolypeptides also have G-Protein binding activity associated with thecytoplasmic domains (including the cytoplasmic tail domain) ofPresenilin polypeptides. Thus, for uses requiring G-protein activationor binding Presenilin polypeptides of the disclosure will include thosehaving a cytoplasmic tail domain and exhibiting G-protein bindingactivity. Presenilin polypeptides of the disclosure further includeoligomers or fusion polypeptides comprising at least one extracellularloop domain and/or cytoplasmic tail domain of one or more Presenilinpolypeptides of the disclosure, and fragments of any of thesepolypeptides that have heterotypic binding and/or G-protein domainbinding activity. The binding activity or activities of humanPresenilin-1 and species homologues and other Presenilin familypolypeptides may be determined, for example, in a yeast two-hybridassay, or in an in vitro assay that measures binding between aPresenilin polypeptide and one β-APP and/or G-protein domain-containingbinding partners, where either the Presenilin polypeptide or its bindingpartner is labeled with a radioactive, fluorescent, or bioluminescentprotein such that binding can be detected.

The term “human Presenilin polypeptide activity,” as used herein,includes β-APP binding and interactions and G-protein binding oractivation. The degree to which Presenilin polypeptides of thedisclosure and fragments and other derivatives of these polypeptidesexhibit these activities can be determined by standard assay methods.Exemplary assays are disclosed herein; those of skill in the art willappreciate that other, similar types of assays can be used to measurethe biological activities of Presenilin polypeptides of the disclosureand other Presenilin family members.

One aspect of the biological activity of Presenilin polypeptidesincluding human Presenilin-1 is the ability of members of thispolypeptide family to bind particular binding partners such heterotypicpolypeptides (including β-APP) and G-protein domain-containingpolypeptides, with the extracellular loop domains binding, for example,to β-APP, and the cytoplasmic tail domain binding to G-proteindomain-containing polypeptides. The term “binding partner,” as usedherein, includes ligands, receptors, substrates, antibodies, otherPresenilin polypeptides, and any other molecule that interacts with ahuman Presenilin-1 polypeptide through contact or proximity betweenparticular portions of the binding partner and the human Presenilin-1 or-2 polypeptide. Because the extracellular N-terminal and loop domains ofPresenilin polypeptides of the disclosure bind to heterotypicpolypeptides, a derivative polypeptide comprising f fragment of theN-terminal 80 amino acids and/or one or more extracellular loop domainswhen expressed as a separate fragment from the rest of a humanPresenilin-1 polypeptide, or as a soluble polypeptide, fused for exampleto an immunoglobulin Fc domain, is expected to disrupt the binding ofPresenilin polypeptides of the disclosure to its binding partners (e.g.,β-APP). By binding to one or more binding partners, the separateextracellular domain(s) polypeptide prevents binding by the native humanPresenilin-1 polypeptide(s), and so acts in a dominant negative fashionto inhibit the biological activities mediated via binding of Presenilinpolypeptides of the disclosure heterotypic polypeptides (e.g., β-APP),thereby, in one aspect, preventing the formation of A. The biologicalactivities and partner-binding properties of human Presenilin-1 andother Presenilin family polypeptides may be assayed by standard methodsand by those assays described herein.

As described herein, Presenilin-1 and -2 has been shown GPCRs relatedtransmembrane proteins that, when activated cause production of Aβpeptides. Therefore, Presenilin-1 and -2 are involved in conditions anddisorders Alzheimer's Disease development and disorders related thereto,including memory modification. Blocking or inhibiting the interactionsbetween Presenilin polypeptides of the disclosure and their substrates,ligands, receptors, binding partners, and or other interactingpolypeptides is an aspect of the disclosure and provides methods fortreating or ameliorating these diseases and conditions through the useof inhibitors of human Presenilin-1 and -2 activity. Examples of suchinhibitors or antagonists are described in more detail below.

In one embodiment, a Presenilin-1 or -2 polypeptide or polynucleotideplays a role normal memory and Alzheimer's Disease development andprogression. In one embodiment, the methods and compositions of thedisclosure include antagonists of Presenilin-1 or -2 activity comprisinga peptide, peptidomimetic, small molecule or other agent the preventsthe interaction of a Presenilin with β-App or activation of a G-protein.

A human Presenilin-1 polypeptide is a polypeptide that (a) has asequence as set forth in SEQ ID NO:2; (b) shares a sufficient degree ofamino acid identity or similarity to a Presenilin-1 polypeptidecomprising an amino acid sequence as set forth in SEQ ID NO:2; (c) isidentified by those of skill in the art as a polypeptide likely to shareparticular structural domains with a Presenilin-1 polypeptide of SEQ IDNO:2; (d) has biological activities in common with a Presenilinpolypeptide; and/or (e) binds to antibodies that also specifically bindto a Presenilin-1 polypeptide having a sequence as set forth in SEQ IDNO:2. A human Presenilin-2 polypeptide is a polypeptide that (a) has asequence as set forth in SEQ ID NO:4; (b) shares a sufficient degree ofamino acid identity or similarity to a Presenilin-2 polypeptidecomprising an amino acid sequence as set forth in SEQ ID NO:4; (c) isidentified by those of skill in the art as a polypeptide likely to shareparticular structural domains with a Presenilin-2 polypeptide of SEQ IDNO:4; (d) has biological activities in common with a Presenilinpolypeptide; and/or (e) binds to antibodies that also specifically bindto a Presenilin-2 polypeptide having a sequence as set forth in SEQ IDNO:4. Presenilin polypeptides of the disclosure may be isolated fromnaturally occurring sources, or be recombinantly produced such that arecombinant Presenilin polypeptide has the same structure as naturallyoccurring Presenilin polypeptides, or may be produced to have structuresthat differ from naturally occurring Presenilin polypeptides.Polypeptides derived from any human Presenilin-1 or -2 polypeptide byany type of alteration (for example, but not limited to, insertions,deletions, or substitutions of, for example, 1-10 or more amino acids;changes in the state of glycosylation of the polypeptide; refolding orisomerization to change its three-dimensional structure orself-association state; and changes to its association with otherpolypeptides or molecules) are also Presenilin polypeptides of thedisclosure. Therefore, the polypeptides provided by the disclosureinclude polypeptides characterized by amino acid sequences similar tothose of the Presenilin polypeptides of the disclosure described herein,but into which modifications are naturally provided or deliberatelyengineered. A polypeptide that shares biological activities in commonwith Presenilin polypeptides of the disclosure is a polypeptide havingPresenilin-1 activity. Examples of biological activities exhibited bymembers of the Presenilin polypeptide family include, withoutlimitation, β-APP and G-protein activation.

An isolated polypeptide or peptide refers to a molecule comprising asequence of amino acids and which may have, in addition to said aminoacid sequence, additional material covalently linked to either or bothends of the polypeptide or peptide, said additional material between1-10, 10-20, 20-30 or 40-50 additional amino acids covalently linked toeither end, each end, or both ends of polypeptide and which polypeptideis removed from its natural state or is recombinantly produced usingmolecular biology or peptide synthesis techniques.

The disclosure provides both full-length and mature forms of Presenilinpolypeptides of the disclosure. Full-length polypeptides are thosehaving the complete primary amino acid sequence of the polypeptide asinitially translated. The amino acid sequences of full-lengthpolypeptides can be obtained, for example, by translation of thecomplete open reading frame (“ORF”) of a cDNA molecule. Severalfull-length polypeptides may be encoded by a single genetic locus ifmultiple mRNA forms are produced from that locus by alternative splicingor by the use of multiple translation initiation sites. The “matureform” of a polypeptide refers to a polypeptide that has undergonepost-translational processing steps such as cleavage of the signalsequence or proteolytic cleavage to remove a prodomain. Multiple matureforms of a particular full-length polypeptide may be produced, forexample by cleavage of the signal sequence at multiple sites, or bydifferential regulation of proteases that cleave the polypeptide. Themature form(s) of such polypeptide may be obtained by expression, in asuitable mammalian cell or other host cell, of a polynucleotide moleculethat encodes the full-length polypeptide. The sequence of the matureform of the polypeptide may also be determinable from the amino acidsequence of the full-length form, through identification of signalsequences or protease cleavage sites. The Presenilin polypeptides of thedisclosure also include those that result from post-transcriptional orpost-translational processing events such as alternate mRNA processingwhich can yield a truncated but biologically active polypeptide, forexample, a naturally occurring soluble form of the polypeptide. Alsoencompassed within the disclosure are variations attributable toproteolysis such as differences in the N- or C-termini upon expressionin different types of host cells, due to proteolytic removal of one ormore terminal amino acids from the polypeptide (generally from 1 to 5terminal amino acids).

The disclosure further includes Presenilin polypeptides of thedisclosure with or without associated native-pattern glycosylation.Polypeptides expressed in yeast or mammalian expression systems (e.g.,COS-1 or CHO cells) can be similar to or significantly different from anative polypeptide in molecular weight and glycosylation pattern,depending upon the choice of expression system. Expression ofpolypeptides of the disclosure in bacterial expression systems, such asE. coli, provides non-glycosylated molecules. Further, a givenpreparation can include multiple differentially glycosylated species ofthe polypeptide. Glycosyl groups can be removed through conventionalmethods, in particular those utilizing glycopeptidase. In general,glycosylated polypeptides of the disclosure can be incubated with amolar excess of glycopeptidase (Boehringer Mannheim).

Species homologues of Presenilin polypeptides of the disclosure (e.g.,the Presenilin-1 human and murine forms) and of polynucleotides encodingthem are encompassed by the disclosure. As used herein, a “specieshomologue” is a polypeptide or polynucleotide with a different speciesof origin from that of a given polypeptide or polynucleotide, but withsignificant sequence similarity to the given polypeptide orpolynucleotide, as determined by those of skill in the art. Specieshomologues may be isolated and identified by making suitable probes orprimers from polynucleotides encoding the amino acid sequences providedherein and screening a suitable nucleic acid source from the desiredspecies. The disclosure also encompasses allelic variants of Presenilinpolypeptides of the disclosure and polynucleotides encoding them; thatis, naturally-occurring alternative forms of such polypeptides andpolynucleotides in which differences in amino acid or nucleotidesequence are attributable to genetic polymorphism (allelic variationamong individuals within a population).

Fragments of the Presenilin polypeptides of the disclosure may be inlinear form or cyclized using known methods, for example, as describedin H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, et al., J. Amer. Chem. Soc. 114 9245-9253 (1992), both ofwhich are incorporated by reference herein. Polypeptides and polypeptidefragments of the disclosure, and polynucleotides encoding them, includepolypeptides and polynucleotides with amino acid or nucleotide sequencelengths that are at least 25% (e.g., at least 50%, or at least 60%, orat least 70%, or at least 80%) of the length of a Presenilin-1polypeptide and have at least 60% sequence identity (e.g., at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97.5%, or at least 99%, or at least 99.5%) with a Presenilin-1polypeptide or encoding polynucleotide, where sequence identity isdetermined by comparing the amino acid sequences of the polypeptideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. Also included in the disclosure are polypeptides andpolypeptide fragments, and polynucleotides encoding them, that containor encode a segment typically comprising at least 8, or at least 10, orat least 15, or at least 20, or at least 30, or at least 40 contiguousamino acids. Such polypeptides and polypeptide fragments may alsocontain a segment that shares at least 70% sequence identity (or atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97.5%, at least 99%, or at least 99.5%) with anysuch segment of any of the Presenilin polypeptides of the disclosure,where sequence identity is determined by comparing the amino acidsequences of the polypeptides when aligned so as to maximize overlap andidentity while minimizing sequence gaps. The percent identity can bedetermined by visual inspection and mathematical calculation.Alternatively, the percent identity of two amino acid or twopolynucleotide sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The typicaldefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Polypeptide Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by those skilled inthe art of sequence comparison may also be used, such as, for example,the BLASTN program version 2.0.9, available for use via the NationalLibrary of Medicine website ncbi.nlm.nih.gov/gorf/wblast2.cgi, or theUW-BLAST 2.0 algorithm. Standard default parameter settings for UW-BLAST2.0 are described at the following Internet webpage:blast.wustl.edu/blast/README.html#References. In addition, the BLASTalgorithm uses the BLOSUM64 amino acid scoring matrix, and optionalparameters that may be used are as follows: (A) inclusion of a filter tomask segments of the query sequence that have low compositionalcomplexity (as determined by the SEG program of Wootton & Federhen(Computers and Chemistry, 1993); also see Wootton J C and Federhen S,1996, Analysis of compositionally biased regions in sequence databases,Methods Enzymol. 266: 554-71) or segments consisting ofshort-periodicity internal repeats (as determined by the XNU program ofClaverie & States (Computers and Chemistry, 1993)), and (B) astatistical significance threshold for reporting matches againstdatabase sequences, or E-score (the expected probability of matchesbeing found merely by chance, according to the stochastic model ofKarlin and Altschul (1990); if the statistical significance ascribed toa match is greater than this E-score threshold, the match will not bereported.); typical E-score threshold values are 0.5, or 0.25, 0.1,0.05, 0.01, 0.001, 0.0001, 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30,1e-40, 1e-50, 1e-75, or 1e-100.

The disclosure also provides for soluble forms of Presenilinpolypeptides of the disclosure comprising certain fragments or domainsof these polypeptides, and particularly those comprising theextracellular domain or one or more fragments of the extracellulardomain. Soluble polypeptides are polypeptides that are capable of beingsecreted from the cells in which they are expressed. In such forms partor all of the intracellular and transmembrane domains of the polypeptideare deleted such that the polypeptide is fully secreted from the cell inwhich it is expressed. The intracellular and transmembrane domains ofpolypeptides of the disclosure can be identified in accordance withknown techniques for determination of such domains from sequenceinformation. Soluble Presenilin polypeptides of the disclosure alsoinclude those polypeptides which include part of the transmembraneregion, provided that the soluble Presenilin-1 polypeptide is capable ofbeing secreted from a cell, and which typically retains a humanPresenilin-1 activity (e.g., such as the ability to bind to or interactwith a β-APP. Soluble Presenilin polypeptides of the disclosure furtherinclude oligomers or fusion polypeptides comprising the extracellularportion of at least one Presenilin-1 or -2 polypeptide, and fragmentsthat have Presenilin-1 or -2 activity. A secreted soluble polypeptidemay be identified (and distinguished from its non-soluble membrane-boundcounterparts) by separating intact cells which express the desiredpolypeptide from the culture medium, e.g., by centrifugation, andassaying the medium (supernatant) for the presence of the desiredpolypeptide. The presence of the desired polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the polypeptide. Purification of the polypeptides fromrecombinant host cells is facilitated, since the soluble polypeptidesare secreted from the cells. Moreover, soluble polypeptides aregenerally more suitable than membrane-bound forms for parenteraladministration and for many enzymatic procedures.

In another aspect of the disclosure, polypeptides comprise variouscombinations of Presenilin-1 polypeptide domains, such as thecytoplasmic tail domain and the extracellular loop domain or acytoplasmic tail and a cytoplasmic loop domain. Accordingly,polypeptides of the disclosure and polynucleotides encoding them includethose comprising or encoding two or more copies of a domain such as thecytoplasmic tail domain, two or more copies of a domain such as theextracellular loop domain, or at least one copy of each domain, andthese domains may be presented in any order within such polypeptides.

Further modifications in the peptide or DNA sequences can be made bythose skilled in the art using known techniques. Modifications ofinterest in the polypeptide sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid. For example, one or more of the cysteine residues may be deletedor replaced with another amino acid to alter the conformation of themolecule, an alteration which may involve preventing formation ofincorrect intramolecular disulfide bridges upon folding or renaturation.Techniques for such alteration, substitution, replacement, insertion ordeletion are well known to those skilled in the art (see, e.g., U.S.Pat. No. 4,518,584). As another example, N-glycosylation sites in thepolypeptide extracellular domain can be modified to precludeglycosylation, allowing expression of a reduced carbohydrate analog inmammalian and yeast expression systems. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.Appropriate substitutions, additions, or deletions to the nucleotidesequence encoding these triplets will result in prevention of attachmentof carbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site.Alternatively, the Ser or Thr can be replaced with another amino acid,such as Ala. Known procedures for inactivating N-glycosylation sites inpolypeptides include those described in U.S. Pat. No. 5,071,972 and EP276,846, hereby incorporated by reference. Additional variants withinthe scope of the disclosure include polypeptides that can be modified tocreate derivatives thereof by forming covalent or aggregative conjugateswith other chemical moieties, such as glycosyl groups, lipids,phosphate, acetyl groups and the like. Covalent derivatives can beprepared by linking the chemical moieties to functional groups on aminoacid side chains or at the N-terminus or C-terminus of a polypeptide.Conjugates comprising diagnostic (detectable) or therapeutic agentsattached thereto are contemplated herein. Preferably, such alteration,substitution, replacement, insertion or deletion retains the desiredactivity of the polypeptide or a substantial equivalent thereof. Oneexample is a variant that binds with essentially the same bindingaffinity as does the native form. Binding affinity can be measured byconventional procedures, e.g., as described in U.S. Pat. No. 5,512,457and as set forth herein.

Other derivatives include covalent or aggregative conjugates of thepolypeptides with other polypeptides or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion polypeptides are discussed below in connection witholigomers. Further, fusion polypeptides can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, which is highly antigenic andprovides an epitope reversibly bound by a specific monoclonal antibody,enabling rapid assay and facile purification of expressed recombinantpolypeptide. A murine hybridoma designated 4E11 produces a monoclonalantibody that binds the FLAG® peptide in the presence of certaindivalent metal cations, as described in U.S. Pat. No. 5,011,912, herebyincorporated by reference. The 4E11 hybridoma cell line has beendeposited with the American Type Culture Collection under accession no.HB 9259. Monoclonal antibodies that bind the FLAG® peptide are availablefrom Eastman Kodak Co., Scientific Imaging Systems Division, New Haven,Conn.

Encompassed by the disclosure are oligomers or fusion polypeptides thatcontain a Presenilin-1 or -2 polypeptide, one or more fragments ofPresenilin polypeptides of the disclosure, or any of the derivative orvariant forms of Presenilin polypeptides of the disclosure as disclosedherein. In particular embodiments, the oligomers comprise solublePresenilin polypeptides of the disclosure. Oligomers can be in the formof covalently linked or non-covalently-linked multimers, includingdimers, trimers, or higher oligomers. In one aspect of the disclosure,the oligomers maintain the binding ability of the polypeptide componentsand provide therefor, bivalent, trivalent, etc., binding sites. In analternative embodiment the disclosure is directed to oligomerscomprising multiple Presenilin polypeptides of the disclosure joined viacovalent or non-covalent interactions between peptide moieties fused tothe polypeptides, such peptides having the property of promotingoligomerization. Leucine zippers and certain polypeptides derived fromantibodies are among the peptides that can promote oligomerization ofthe polypeptides attached thereto, as described in more detail below.

In embodiments where variants of the Presenilin polypeptides of thedisclosure are constructed to include a membrane-spanning domain, theywill form a membrane-spanning polypeptide. Membrane-spanning Presenilinpolypeptides of the disclosure can be fused with extracellular domainsof receptor polypeptides for which the ligand is known. Such fusionpolypeptides can then be manipulated to control the intracellularsignaling pathways triggered by the membrane-spanning Presenilin-1polypeptide. Presenilin polypeptides of the disclosure that span thecell membrane can also be fused with agonists or antagonists ofcell-surface receptors, or cellular adhesion molecules to furthermodulate Presenilin-1 intracellular effects. In another aspect of thedisclosure, interleukins can be situated between Presenilin-1polypeptide fragment and other fusion polypeptide domains.

Immunoglobulin-based Oligomers. The polypeptides of the disclosure orfragments thereof may be fused to molecules such as immunoglobulins formany purposes, including increasing the valency of polypeptide bindingsites. For example, fragments of a Presenilin-1 or -2 polypeptide may be(a) fused directly or through a linker peptide to the Fc portion of animmunoglobulin, or (b) fused directly or through a linker peptide toanother Presenilin-1 polypeptide. For a bivalent form of thepolypeptide, such a fusion could be to the Fc portion of an IgGmolecule. Other immunoglobulin isotypes may also be used to generatesuch fusions. For example, a polypeptide-IgM fusion would generate adecavalent form of the polypeptide of the disclosure. The term “Fcpolypeptide” as used herein includes native and mutein forms ofpolypeptides made up of the Fc region of an antibody comprising any orall of the CH domains of the Fc region. Truncated forms of suchpolypeptides containing the hinge region that promotes dimerization arealso included. Useful Fc polypeptides comprise an Fc polypeptide derivedfrom a human IgG1 antibody. As one alternative, an oligomer is preparedusing polypeptides derived from immunoglobulins. Preparation of fusionpolypeptides comprising certain heterologous polypeptides fused tovarious portions of antibody-derived polypeptides (including the Fcdomain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh andAruffo (“Construction of Immunoglobulin Fusion Polypeptides”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992).Methods for preparation and use of immunoglobulin-based oligomers arewell known in the art. One embodiment of the disclosure is directed to adimer comprising two fusion polypeptides created by fusing a polypeptideof the disclosure to an Fc polypeptide derived from an antibody. A genefusion encoding the polypeptide/Fc fusion polypeptide is inserted intoan appropriate expression vector. Polypeptide/Fc fusion polypeptides areexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yielddivalent molecules. One suitable Fc polypeptide, described in PCTapplication WO 93/10151 (hereby incorporated by reference), is a singlechain polypeptide extending from the N-terminal hinge region to thenative C-terminus of the Fc region of a human IgG1 antibody. Anotheruseful Fc polypeptide is the Fc mutein described in U.S. Pat. No.5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994) incorporatedherein by reference. The amino acid sequence of this mutein is identicalto that of the native Fc sequence presented in WO 93/10151, except thatamino acid 19 has been changed from Leu to Ala, amino acid 20 has beenchanged from Leu to Glu, and amino acid 22 has been changed from Gly toAla. The mutein exhibits reduced affinity for Fc receptors. Theabove-described fusion polypeptides comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Polypeptide A or Polypeptide G columns.In other embodiments, the polypeptides of the disclosure can besubstituted for the variable portion of an antibody heavy or lightchain. If fusion polypeptides are made with both heavy and light chainsof an antibody, it is possible to form an oligomer with as many as fourPresenilin-1 extracellular regions.

Alternatively, the oligomer is a fusion polypeptide comprising multiplePresenilin polypeptides of the disclosure, with or without peptidelinkers (spacer peptides). Among the suitable peptide linkers are thosedescribed in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are herebyincorporated by reference. An oligonucleotide sequence encoding adesired peptide linker can be inserted between, and in the same readingframe as a Presenilin polynucleotide of the disclosure, using anysuitable conventional technique. For example, a chemically synthesizedoligonucleotide encoding a peptide linker can be ligated between thesequences. In particular embodiments, a fusion polypeptide comprisesfrom two to four soluble Presenilin polypeptides of the disclosure,separated by peptide linkers. Suitable peptide linkers, theircombination with other polypeptides, and their use are well known bythose skilled in the art

Another method for preparing the oligomers of the disclosure involvesuse of a leucine zipper. Leucine zipper domains are peptides thatpromote oligomerization of the polypeptides in which they are found.Leucine zippers were originally identified in several DNA-bindingpolypeptides (Landschulz et al., Science 240:1759, 1988), and have sincebeen found in a variety of different polypeptides. Among the knownleucine zippers are naturally occurring peptides and derivatives thereofthat dimerize or trimerize. The zipper domain (also referred to hereinas an oligomerizing, or oligomer-forming, domain) comprises a repetitiveheptad repeat, often with four or five leucine residues interspersedwith other amino acids. Use of leucine zippers and preparation ofoligomers using leucine zippers are well known in the art.

Other fragments and derivatives of the sequences of polypeptides whichwould be expected to retain polypeptide activity in whole or in part andmay thus be useful for screening or other immunological methodologiesmay also be made by those skilled in the art given the disclosuresherein. Such modifications are encompassed by the disclosure.

The disclosure provides soluble peptide fragments useful for treating ADby binding to β-APP and preventing β-APP interaction with the fulllength native Presenilin-1 or -2. Useful fragments include, but are notlimited to: (i) a sequence consisting of N-DEEEDEEL-COOH (SEQ ID NO:5),(ii) a sequence consisting of SEQ ID NO:5 further including 1-50additional amino acids at either the N- or C-terminal end so long as thepeptide is capable of binding to a β-APP, (iii) the sequenceN-RRSLGHPEPLSNGRP-COOH (SEQ ID NO:6), (iv) a sequence consisting of SEQID NO:6 further including 1-5 conservative amino acid substitutions, (v)a sequence consisting of (iii) or (iv) further including 1-50 additionalamino acids at the N- or C-terminus, (vi) the sequenceN-RRSLGHPEPLSNGRPQGNSRQVVEQDEEEDEELTLKYGAK-COOH (SEQ ID NO:7), (vii) asequence consisting of SEQ ID NO:7 further including 1-5 conservativeamino acid substitutions, (viii) a sequence consisting of (vi) or (vii)further including 1-50 additional amino acids at the N- or C-terminus,and (ix) any of the foregoing comprising an unnatural amino acid orD-amino acid so long as the peptide are capable of interacting orbinding to a β-APP. The disclosure demonstrates that PS-1 domains withinthe 80 amino-acid N-terminal fragment can specifically inhibit theproduction of Aβ when added to co-cultures of β-APP and PS-1 expressingcells. These peptides all inhibit Aβ production in β-APP transgenicmice. The disclosure further demonstrates that if cell-cell interactionis inhibited, then both G-protein activation and Aβ production are alsoinhibited.

The disclosure also provides inhibitors of G-protein activation inducedby interaction of β-APP and PS-1 or PS-2. For example, if the G-proteinactivation is inhibited (by the presence of PTx in the β-APP:PS-1co-cultures), then Aβ production is also inhibited. Accordingly, thedisclosure provides the sequence of the PS-1 intracellular domainrequired for G-protein activation and GoA binding. In one embodiment,the disclosure provides a C-terminal tail sequence comprising the first20 amino acids of PS-1 (N-KKALPALPISITFGLVFYFA-COOH; SEQ ID NO:8). Inaddition, the disclosure identifies intracellular loop 3 (KYLPE; SEQ IDNO:2 amino acids 239-243) of PS-1 (which has identity to thecorresponding domain in PS-2) and peptides that binds Go_(A) binding,including for example, N-MALVFIKYLPE-COOH; SEQ ID NO:9.

Encompassed within the disclosure are polynucleotides encoding suchPresenilin polypeptides or peptides of the disclosure. Thesepolynucleotides can be identified in several ways, including isolationof genomic or cDNA molecules from a suitable source. Nucleotidesequences corresponding to the amino acid sequences described herein, tobe used as probes or primers for the isolation of polynucleotides or asquery sequences for database searches, can be obtained by“back-translation” from the amino acid sequences, or by identificationof regions of amino acid identity with polypeptides for which the codingDNA sequence has been identified. The well-known polymerase chainreaction (PCR) procedure can be employed to isolate and amplify a DNAsequence encoding a human Presenilin-1 or -2 polypeptide or a desiredcombination of human Presenilin-1 or -2 polypeptide fragments.Oligonucleotides that define the desired termini of the combination ofDNA fragments are employed as 5′ and 3′ primers. The oligonucleotidescan additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified combination ofDNA fragments into an expression vector. PCR techniques are described inSaiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu etal., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCRProtocols: A Guide to Methods and Applications, Innis et al., eds.,Academic Press, Inc. (1990).

Polynucleotide molecules of the disclosure include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. The polynucleotide molecules of the disclosure includefull-length genes or cDNA molecules as well as a combination offragments thereof. The polynucleotides of the disclosure can be derivedfrom human sources, but the disclosure includes those derived fromnon-human species, as well.

An “isolated polynucleotide” is a polynucleotide that has been separatedfrom adjacent genetic sequences present in the genome of the organismfrom which the polynucleotide was isolated, in the case ofpolynucleotides isolated from naturally occurring sources. In the caseof polynucleotides synthesized enzymatically from a template orchemically, such as PCR products, cDNA molecules, or oligonucleotidesfor example, it is understood that the polynucleotides resulting fromsuch processes are isolated polynucleotides. An isolated polynucleotiderefers to a polynucleotide in the form of a separate fragment or as acomponent of a larger polynucleotide construct. In one embodiment, thedisclosure relates to certain isolated polynucleotides that aresubstantially free from contaminating endogenous material. Thepolynucleotide has preferably been derived from DNA or RNA isolated atleast once in substantially pure form and in a quantity or concentrationenabling identification, manipulation, and recovery of its componentnucleotide sequences by standard biochemical methods (such as thoseoutlined in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).Such sequences are typically provided and/or constructed in the form ofan open reading frame uninterrupted by internal non-translatedsequences, or introns, that are typically present in eukaryotic genes.Sequences of non-translated DNA can be present 5′ or 3′ from an openreading frame, where the same do not interfere with manipulation orexpression of the coding region.

Methods for making Presenilin polypeptides of the disclosure aredescribed below. Expression, isolation, and purification of thepolypeptides and fragments of the disclosure can be accomplished by anysuitable technique, including but not limited to, the following methods.The isolated nucleic acid of the disclosure can be operably linked to anexpression control sequence such as the pDC409 vector (Giri et al.,1990, EMBO J. 13: 2821) or the derivative pDC412 vector (Wiley et al.,1995, Immunity 3: 673). The pDC400 series vectors are useful fortransient mammalian expression systems, such as CV-1 or 293 cells.Alternatively, the isolated nucleic acid of the disclosure can be linkedto expression vectors such as pDC312, pDC316, or pDC317 vectors. ThepDC300 series vectors all contain the SV40 origin of replication, theCMV promoter, the adenovirus tripartite leader, and the SV40 polyA andtermination signals, and are useful for stable mammalian expressionsystems, such as CHO cells or their derivatives. Other expressioncontrol sequences and cloning technologies can also be used to producethe polypeptide recombinantly, such as the pMT2 or pED expressionvectors (Kaufman et al., 1991, Nucleic Acids Res 19: 4485-4490; andPouwels et al., 1985, Cloning Vectors. A Laboratory Manual, Elsevier,New York) and the GATEWAY Vectors (Life Technologies; Rockville, Md.).The isolated nucleic acid of the disclosure, flanked by attB sequences,can be recombined through an integrase reaction with a GATEWAY vectorsuch as pDONR201 containing attP sequences, providing an entry vectorfor the GATEWAY system containing the isolated nucleic acid of thedisclosure. This entry vector can be further recombined with othersuitably prepared expression control sequences, such as those of thepDC400 and pDC300 series described above. Many suitable expressioncontrol sequences are known in the art. General methods of expressingrecombinant polypeptides are also described in Kaufman, 1990, Methods inEnzymology 185, 537-566. As used herein “operably linked” means that apolynucleotide of the disclosure and an expression control sequence aresituated within a construct, vector, or cell in such a way that apolypeptide encoded by a polynucleotide is expressed when appropriatemolecules (such as polymerases) are present. As one embodiment of thedisclosure, at least one expression control sequence is operably linkedto a polynucleotide of the disclosure in a recombinant host cell orprogeny thereof, the polynucleotide and/or expression control sequencehaving been introduced into the host cell by transformation ortransfection, for example, or by any other suitable method. As anotherembodiment of the disclosure, at least one expression control sequenceis integrated into the genome of a recombinant host cell such that it isoperably linked to a polynucleotide sequence encoding a polypeptide ofthe disclosure. In a further embodiment of the disclosure, at least oneexpression control sequence is operably linked to a polynucleotide ofthe disclosure through the action of a trans-acting factor such as atranscription factor, either in vitro or in a recombinant host cell.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. The choiceof signal peptide or leader can depend on factors such as the type ofhost cells in which the recombinant polypeptide is to be produced. Toillustrate, examples of heterologous signal peptides that are functionalin mammalian host cells include the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846. A DNA sequence for a signal peptide(secretory leader) can be fused in frame to a polynucleotide of thedisclosure so that the DNA is initially transcribed, and the mRNAtranslated, into a fusion polypeptide comprising the signal peptide. Asignal peptide that is functional in the intended host cells promotesextracellular secretion of the polypeptide. The signal peptide iscleaved from the polypeptide upon secretion of polypeptide from thecell. The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved can differ from that predicted bycomputer program, and can vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. Apolypeptide preparation can include a mixture of polypeptide moleculeshaving different N-terminal amino acids, resulting from cleavage of thesignal peptide at more than one site.

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, 1990, Large Scale Mammalian Cell Culture, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipidreagent, can be used to transfect cells (Felgner et al., 1987, Proc.Natl. Acad. Sci. USA 84: 7413-7417). In addition, electroporation can beused to transfect mammalian cells using conventional procedures, such asthose in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed.Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection ofstable transformants can be performed using methods known in the artsuch as, for example, resistance to cytotoxic drugs. Kaufman et al.,Meth. in Enzymology 185:487-511, 1990, describes several selectionschemes, such as dihydrofolate reductase (DHFR) resistance. A suitablestrain for DHFR selection can be CHO strain DX-B11, which is deficientin DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220,1980). A plasmid expressing the DHFR cDNA can be introduced into strainDX-B11, and only cells that contain the plasmid can grow in theappropriate selective media. Other examples of selectable markers thatcan be incorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Alternatively, gene products can be obtained via homologousrecombination, or “gene targeting,” techniques. Such techniques employthe introduction of exogenous transcription control elements (such asthe CMV promoter or the like) in a particular predetermined site on thegenome, to induce expression of the endogenous polynucleotide sequenceof interest. The location of integration into a host chromosome orgenome can be easily determined by one of skill in the art, given theknown location and sequence of the gene. In one embodiment, thedisclosure also contemplates the introduction of exogenoustranscriptional control elements in conjunction with an amplifiablegene, to produce increased amounts of the gene product, again, withoutthe need for isolation of the gene itself from the host cell. Thepractice of homologous recombination or gene targeting is explained bySchimke, et al. “Amplification of Genes in Somatic Mammalian cells,”Methods in Enzymology 151:85-104 (1987), as well as by Capecchi, et al.,“The New Mouse Genetics. Altering the Genome by Gene Targeting,” TIG5:70-76 (1989).

A number of types of cells may act as suitable host cells for expressionof a polypeptide. Mammalian host cells include, for example, the COS-7line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, theCV1/EBNA cell line derived from the African green monkey kidney cellline CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821,1991), human kidney 293 cells, human epidermal A431 cells, human Colo205cells, other transformed primate cell lines, normal diploid cells, cellstrains derived from in vitro culture of primary tissue, primaryexplants, HL-60, U937, HaK or Jurkat cells. Alternatively, it may bepossible to produce a polypeptide in lower eukaryotes such as yeast orin prokaryotes such as bacteria. Potentially suitable yeast strainsinclude Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous polypeptides. Potentially suitable bacterialstrains include Escherichia coli, Bacillus subtilis, Salmonellatyphimurium, or any bacterial strain capable of expressing heterologouspolypeptides. If the polypeptide is made in yeast or bacteria, it may benecessary to modify the polypeptide produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional polypeptide. Such covalent attachments may beaccomplished using known chemical or enzymatic methods. The polypeptidemay also be produced by operably linking an isolated polynucleotide ofthe disclosure to suitable control sequences in one or more insectexpression vectors, and employing an insect expression system. Materialsand methods for baculovirus/insect cell expression systems arecommercially available in kit form from, e.g., Invitrogen, San Diego,Calif., U.S.A. (the MaxBac® kit), and such methods are well known in theart, as described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology6:47 (1988), incorporated herein by reference. As used herein, an insectcell capable of expressing a polynucleotide of the disclosure is“transformed.” Cell-free translation systems could also be employed toproduce polypeptides using RNAs derived from polynucleotide constructsdisclosed herein. A host cell that comprises an isolated polynucleotideof the disclosure, typically operably linked to at least one expressioncontrol sequence, is a “recombinant host cell”.

A polypeptide or peptide of the disclosure may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant polypeptide. The resulting expressed polypeptide may then bepurified from such culture (e.g., from culture medium or cell extracts)using known purification processes, such as gel filtration and ionexchange chromatography. The purification of a polypeptide may alsoinclude an affinity column containing agents which will bind to thepolypeptide; one or more column steps over such affinity resins asconcanavalin A-agarose, Heparin-Toyopearl® or Cibacrom blue 3GASepharose®; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography. Alternatively, a polypeptide ofthe disclosure may also be expressed in a form that will facilitatepurification. For example, it may be expressed as a fusion polypeptide,such as those of maltose binding polypeptide (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion polypeptides are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and InVitrogen, respectively. A polypeptide can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (“Flag”) iscommercially available from Kodak (New Haven, Conn.). Finally, one ormore reverse-phase high performance liquid chromatography (RP-HPLC)steps employing hydrophobic RP-HPLC media, e.g., silica gel havingpendant methyl or other aliphatic groups, can be employed to furtherpurify the polypeptide. Some or all of the foregoing purification steps,in various combinations, can also be employed to provide a substantiallyhomogeneous isolated recombinant polypeptide. A polypeptide thuspurified is substantially free of other mammalian polypeptides and isdefined in accordance with the disclosure as a “purified polypeptide”;such purified polypeptides of the disclosure include purified antibodiesthat bind to Presenilin polypeptides of the disclosure, fragments,variants, binding partner, and the like. A polypeptide of the disclosuremay also be expressed as a product of transgenic animals, e.g., as acomponent of the milk of transgenic cows, goats, pigs, or sheep whichare characterized by somatic or germ cells containing a polynucleotideencoding the polypeptide.

It is also possible to utilize an affinity column comprising apolypeptide-binding polypeptide of the disclosure, such as a monoclonalantibody generated against polypeptides of the disclosure, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the disclosure. In this aspect of the disclosure,polypeptide-binding polypeptides, such as the anti-polypeptideantibodies of the disclosure or other polypeptides that can interactwith a polypeptide of the disclosure, can be bound to a solid phasesupport such as a column chromatography matrix or a similar substratesuitable for identifying, separating, or purifying cells that expresspolypeptides of the disclosure on their surface. Adherence ofpolypeptide-binding polypeptides of the disclosure to a solid phasecontacting surface can be accomplished by any number of techniques, forexample, magnetic microspheres can be coated with thesepolypeptide-binding polypeptides and held in the incubation vesselthrough a magnetic field. Suspensions of cell mixtures are contactedwith the solid phase that has such polypeptide-binding polypeptidesthereon. Cells having polypeptides of the disclosure on their surfacebind to the fixed polypeptide-binding polypeptide and unbound cells thenare washed away. This affinity-binding method is useful for purifying,screening, or separating such polypeptide-expressing cells fromsolution. Methods of releasing positively selected cells from the solidphase are known in the art and encompass, for example, the use ofenzymes. Such enzymes are preferably non-toxic and non-injurious to thecells and are directed to cleaving the cell-surface binding partner.Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the disclosure can first be incubatedwith a biotinylated polypeptide-binding polypeptide of the disclosure.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the disclosure. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art (see, e.g., Berenson, et al. J. Cell. Biochem.,10D:239, 1986). Wash of unbound material and the release of the boundcells is performed using conventional methods

A polypeptide may also be produced by known conventional chemicalsynthesis. Methods for constructing polypeptides of the disclosure bysynthetic means are known to those skilled in the art. The syntheticallyconstructed polypeptides, by virtue of sharing primary, secondary ortertiary structural and/or conformational characteristics with nativepolypeptides may possess biological properties in common therewith,including polypeptide activity. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedpolypeptides in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The desired degree of purity depends on the intended use of apolypeptide. A relatively high degree of purity is desired when apolypeptide is to be administered in vivo, for example. In such a case,polypeptides are purified such that no polypeptide bands correspondingto other polypeptides are detectable upon analysis by SDS-polyacrylamidegel electrophoresis (SDS-PAGE). It will be recognized by one skilled inthe pertinent field that multiple bands corresponding to the polypeptidecan be visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. A polypeptideof the disclosure is purified to substantial homogeneity, as indicatedby a single polypeptide band upon analysis by SDS-PAGE. The polypeptideband can be visualized by silver staining, Coomassie blue staining, or(if the polypeptide is radiolabeled) by autoradiography.

Any method that neutralizes Presenilin polypeptides of the disclosure orinhibits expression of a Presenilin-1 or -2 gene (either transcriptionor translation) or which inhibits the interaction of a Presenilin withβ-APP can be used to modify memory and/or the onset or progression ofAlzheimer's Disease. In particular embodiments, antagonists inhibit thebinding of at least one Presenilin-1 polypeptide to binding partnersexpressed on cells, thereby inhibiting biological activities induced bythe binding of those Presenilin polypeptides of the disclosure to thecells. In certain other embodiments of the disclosure, antagonists canbe designed to reduce the level of endogenous Presenilin-1 or 2 geneexpression, e.g., using well-known antisense or ribozyme approaches toinhibit or prevent translation of Presenilin-1 or -2 mRNA transcripts;triple helix approaches to inhibit transcription of Presenilin-1 genes;or targeted homologous recombination to inactivate or “knock out” aPresenilin-1 or -2 gene or their endogenous promoters or enhancerelements. Such antisense, ribozyme, and triple helix antagonists may bedesigned to reduce or inhibit either unimpaired, or if appropriate,mutant Presenilin-1 or -2 gene activity. Techniques for the productionand use of such molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation ofmRNA by hybridizing to targeted mRNA and preventing polypeptidetranslation. Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to a Presenilin-1 mRNA. Theantisense oligonucleotides will bind to the complementary target genemRNA transcripts and prevent translation. Absolute complementarity,although preferred, is not required. A sequence “complementary” to aportion of a polynucleotide, as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with thepolynucleotide, forming a stable duplex (or triplex, as appropriate). Inthe case of double-stranded antisense nucleic acids, a single strand ofthe duplex DNA may thus be tested, or triplex formation may be assayed.The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid.Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, oligonucleotides complementary to either the 5′-or 3′-non-translated, non-coding regions of a Presenilin-1 or -2 genetranscript could be used in an antisense approach to inhibit translationof endogenous Presenilin-1 or -2 mRNA. Oligonucleotides complementary tothe 5′ untranslated region of the mRNA should include the complement ofthe AUG start codon. Antisense nucleic acids should be at least sixnucleotides in length, and typically range from 6 to about 50nucleotides in length. In specific aspects the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotidesor at least 50 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, and the like. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.,1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810,published Dec. 15, 1988), or hybridization-triggered cleavage agents orintercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). Theantisense molecules should be delivered to cells that express a humanPresenilin-1 or -2 transcript in vivo. A number of methods have beendeveloped for delivering antisense DNA or RNA to cells; e.g., antisensemolecules can be injected directly into the tissue or cell derivationsite, or modified antisense molecules, designed to target the desiredcells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systemically. However, it is oftendifficult to achieve intracellular concentrations of the antisensemolecule sufficient to suppress translation of endogenous mRNAs.Therefore one approach utilizes a recombinant DNA construct in which theantisense oligonucleotide is placed under the control of a strong polIII or pol II promoter. The use of such a construct to transfect targetcells in a subject will result in the transcription of sufficientamounts of single stranded RNAs that will form complementary base pairswith the endogenous Presenilin-1 gene transcripts and thereby preventtranslation of the Presenilin-1 or -2 mRNA. For example, a vector can beintroduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, so long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art used for replication andexpression in mammalian cells.

Ribozyme molecules designed to catalytically cleave Presenilin-1 or -2mRNA transcripts can also be used to prevent translation of Presenilin-1or -2 mRNA thereby inhibiting expression of Presenilin polypeptides ofthe disclosure (see, e.g., PCT International Publication WO90/11364,published Oct. 4, 1990; U.S. Pat. No. 5,824,519). The ribozymes that canbe used in the disclosure include hammerhead ribozymes (Haseloff andGerlach, 1988, Nature, 334:585-591), RNA endoribonucleases (hereinafter“Cech-type ribozymes”) such as the one which occurs naturally inTetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and whichhas been extensively described by Thomas Cech and collaborators(International Patent Application No. WO 88/04300; Been and Cech, 1986,Cell, 47:207-216). As in the antisense approach, the ribozymes can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, and the like) and should be delivered to cells which expressthe human Presenilin-1 polypeptide in vivo. A typical method of deliveryinvolves using a DNA construct coding for the ribozyme under the controlof a strong constitutive pol II or pol III promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous Presenilin-1 or -2 messages and inhibit translation. Becauseribozymes, unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Alternatively, endogenous Presenilin-1 or -2 gene expression can bereduced by targeting deoxyribonucleotide sequences complementary to theregulatory region of the target gene (e.g., the target gene's promoterand/or enhancers) to form triple helical structures that preventtranscription of a Presenilin-1 gene (see generally, Helene, 1991,Anticancer Drug Des., 6(6):569-584; Helene, et al., 1992, Ann. N.Y.Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12):807-815).

Antisense nucleic acids, ribozyme, and triple helix molecules of thedisclosure may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as, for example, solidphase phosphoramidite chemical synthesis. Oligonucleotides can besynthesized by standard methods known in the art, e.g. by use of anautomated DNA synthesizer (such as are commercially available fromBiosearch, Applied Biosystems, and the like). As examples,phosphorothioate oligonucleotides may be synthesized by the method ofStein et al., 1988, Nucl. Acids Res. 16:3209. Methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:7448-7451). Alternatively, RNA molecules may be generated by in vitroand in vivo transcription of DNA sequences encoding the antisense RNAmolecule. Such DNA sequences may be incorporated into a wide variety ofvectors that incorporate suitable RNA polymerase promoters such as theT7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructsthat synthesize antisense RNA constitutively or inducibly, depending onthe promoter used, can be introduced stably into cell lines.

Organisms that have enhanced, reduced, or modified expression of thegene(s) corresponding to the polynucleotide sequences disclosed hereinare provided. The desired change in gene expression can be achievedthrough the use of antisense nucleic acids or ribozymes that bind and/orcleave the mRNA transcribed from the gene (Albert and Morris, 1994,Trends Pharmacol. Sci. 15(7):250-254; Lavarosky et al., 1997, Biochem.Mol. Med. 62(1):11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol.Biol. 58:1-39; all of which are incorporated by reference herein).Transgenic animals that have multiple copies of the gene(s)corresponding to the polynucleotide sequences disclosed herein, producedby transformation of cells with genetic constructs that are stablymaintained within the transformed cells and their progeny, are provided.Transgenic animals that have modified genetic control regions thatincrease or reduce gene expression levels, or that change temporal orspatial patterns of gene expression, are also provided (see, e.g.,European Patent No. 0 649 464 B1, incorporated by reference herein). Inaddition, organisms are provided in which the gene(s) corresponding tothe polynucleotide sequences disclosed herein have been partially orcompletely inactivated, through insertion of extraneous sequences intothe corresponding gene(s) or through deletion of all or part of thecorresponding gene(s). Partial or complete gene inactivation can beaccomplished through insertion, followed by imprecise excision, oftransposable elements (Plasterk, 1992, Bioessays 14(9):629-633; Zwaal etal., 1993, Proc. Natl. Acad. Sci. USA 90(16):7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci. USA 91(2):719-722; all of which areincorporated by reference herein), or through homologous recombinationwhich can be detected by positive/negative genetic selection strategies(Mansour et al., 1988, Nature 336:348-352; U.S. Pat. Nos. 5,464,764;5,487,992; 5,627,059; 5,631,153; 5,614,396; 5,616,491; and 5,679,523;all of which are incorporated by reference herein). These organisms withaltered gene expression are eukaryotes and typically are mammals. Suchorganisms are useful for the development of non-human models for thestudy of disorders involving the corresponding gene(s), and for thedevelopment of assay systems for the identification of molecules thatinteract with the polypeptide product(s) of the corresponding gene(s).

The Presenilin polypeptides of the disclosure themselves can also beemployed in inhibiting a biological activity of Presenilin-1 or -2 in invitro or in vivo procedures. Encompassed within the disclosure areextracellular loop domains of Presenilin polypeptides of the disclosurethat act as “dominant negative” inhibitors of native Presenilin-1 or -2polypeptide function when expressed as fragments or as components offusion polypeptides. For example, a purified polypeptide domain of thedisclosure can be used to inhibit binding of Presenilin polypeptides ofthe disclosure to endogenous binding partners. Such use wouldeffectively block Presenilin-1 or -2 polypeptide interactions with β-APPand inhibit Presenilin-1 or -2 polypeptide activities. In still anotheraspect of the disclosure, a soluble form of a Presenilin-1 or -2 bindingpartner, which is expressed on epithelial and/or endothelial cells, isused to bind to and competitively inhibit activation of an endogenousPresenilin-1 or -2 polypeptide. Furthermore, antibodies which bind toPresenilin polypeptides of the disclosure can inhibit Presenilin-1 or -2activity and act as antagonists, or as agonists. For example, antibodiesthat specifically recognize one or more epitopes of Presenilinpolypeptides of the disclosure, or epitopes of conserved variants ofPresenilin polypeptides of the disclosure, or peptide fragments of aPresenilin-1 polypeptide can be used in the disclosure to inhibitPresenilin-1 or -2 activity (e.g., antagonistic antibodies). Agonisticantibodies bind to Presenilin polypeptides of the disclosure or bindingpartners and increase Presenilin-1 or -2 polypeptide activity by causingconstitutive intracellular signaling (or “ligand mimicking”), or bypreventing the binding of a native inhibitor of Presenilin-1 or -2polypeptide activity. Antibodies which bind to Presenilin-1 or -2polypeptides include, but are not limited to, polyclonal antibodies,monoclonal antibodies (mAbs), human (also called “fully human”)antibodies, humanized or chimeric antibodies, single chain antibodies,Fab fragments, F(ab′)2 fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above. Alternatively, purified and modifiedPresenilin polypeptides of the disclosure can be administered tomodulate interactions between Presenilin polypeptides of the disclosureand Presenilin-1 or -2 binding partners that are not membrane-bound.Such an approach will allow an alternative method for the modificationof human Presenilin-1-influenced bioactivity.

Polypeptides of the disclosure may be used to identify antagonists andagonists from cells, cell-free preparations, chemical libraries, andnatural product mixtures. The antagonists and agonists may be natural ormodified substrates, ligands, enzymes, receptors, etc. of thepolypeptides of the instant disclosure, or may be structural orfunctional mimetics of the polypeptides. Potential antagonists of theinstant disclosure may include small molecules, peptides and antibodiesthat bind to and occupy a binding site of the inventive polypeptides ora binding partner thereof, causing them to be unavailable to bind totheir natural binding partners and therefore preventing normalbiological activity. Potential agonists include small molecules,peptides and antibodies which bind to the instant polypeptides orbinding partners thereof, and elicit the same or enhanced biologiceffects as those caused by the binding of the polypeptides of theinstant disclosure. Peptide agonists and antagonists of the polypeptidesof the disclosure can be identified and utilized according to knownmethods (see, for example, WO 00/24782 and WO 01/83525, which areincorporated by reference herein).

An approach to development of therapeutic agents is peptide libraryscreening. The interaction of a protein ligand with its receptor oftentakes place at a relatively large interface. However, as demonstratedfor human growth hormone and its receptor, only a few key residues atthe interface contribute to most of the binding energy (Clackson et al.,1995; Science 267: 383-386). The bulk of the protein ligand merelydisplays the binding epitopes in the right topology or serves functionsunrelated to binding. Thus, molecules of only “peptide” length (2 to 90amino acids) can bind to the receptor protein or binding partner of evena large protein ligand such as a polypeptide of the disclosure. Suchpeptides may mimic the bioactivity of the large protein ligand (“peptideagonists”) or, through competitive binding, inhibit the bioactivity ofthe large protein ligand (“peptide antagonists”). Exemplary peptideagonists and antagonists of polypeptides of the disclosure may comprisea domain of a naturally occurring molecule or may comprise randomizedsequences. The term “randomized” as used to refer to peptide sequencesrefers to fully random sequences (e.g., selected by phage displaymethods or RNA-peptide screening) and sequences in which one or moreresidues of a naturally occurring molecule is replaced by an amino acidresidue not appearing in that position in the naturally occurringmolecule. Phage display peptide libraries have emerged as a powerfulmethod in identifying such peptide agonists and antagonists. See, forexample, Scott et al., 1990, Science 249: 386; Devlin et al., 1990,Science 249: 404; U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,733,731; U.S.Pat. No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No. 5,338,665;U.S. Pat. No. 5,922,545; WO 96/40987; and WO 98/15833 (each of which isincorporated by reference in its entirety). In such libraries, randompeptide sequences are displayed by fusion with coat proteins offilamentous phage. Typically, the displayed peptides are affinity-elutedagainst an antibody-immobilized extracellular domain of a receptor. Theretained phages may be enriched by successive rounds of affinitypurification and repropagation. The best binding peptides may besequenced to identify key residues within one or more structurallyrelated families of peptides. The peptide sequences may also suggestwhich residues may be safely replaced by alanine scanning or bymutagenesis at the DNA level. Mutagenesis libraries may be created andscreened to further optimize the sequence of the best binders (Lowman,1997, Ann. Rev. Biophys. Biomol. Struct. 26: 401-424). Anotherbiological approach to screening soluble peptide mixtures uses yeast forexpression and secretion (Smith et al., 1993, Mol. Pharmacol. 43:741-748) to search for peptides with favorable therapeutic properties.Hereinafter, this and related methods are referred to as “yeast-basedscreening.” A peptide library can also be fused to the carboxyl terminusof the lac repressor and expressed in E. coli. Another E. coli-basedmethod allows display on the cell's outer membrane by fusion with apeptidoglycan-associated lipoprotein (PAL). Hereinafter, these andrelated methods are collectively referred to as “E. coli display.” Inanother method, translation of random RNA is halted prior to ribosomerelease, resulting in a library of polypeptides with their associatedRNA still attached. Hereinafter, this and related methods arecollectively referred to as “ribosome display.” Other methods employpeptides linked to RNA; for example, PROfusion technology, Phylos, Inc.(see, for example, Roberts and Szostak, 1997, Proc. Natl. Acad. Sci. USA94: 12297-12303). Hereinafter, this and related methods are collectivelyreferred to as “RNA-peptide screening.” Chemically derived peptidelibraries have been developed in which peptides are immobilized onstable, non-biological materials, such as polyethylene rods orsolvent-permeable resins. Another chemically derived peptide libraryuses photolithography to scan peptides immobilized on glass slides.Hereinafter, these and related methods are collectively referred to as“chemical-peptide screening.” Chemical-peptide screening may beadvantageous in that it allows use of D-amino acids and other unnaturalanalogues, as well as non-peptide elements. Both biological and chemicalmethods are reviewed in Wells and Lowman, 1992, Curr. Opin. Biotechnol.3: 355-362.

In the case of known bioactive peptides, rational design of peptideligands with favorable therapeutic properties can be completed. In suchan approach, one makes stepwise changes to a peptide sequence anddetermines the effect of the substitution upon bioactivity or apredictive biophysical property of the peptide (e.g., solutionstructure). Hereinafter, these techniques are collectively referred toas “rational design.” In one such technique, one makes a series ofpeptides in which one replaces a single residue at a time with alanine.This technique is commonly referred to as an “alanine walk” or an“alanine scan.” When two residues (contiguous or spaced apart) arereplaced, it is referred to as a “double alanine walk.” The resultantamino acid substitutions can be used alone or in combination to resultin a new peptide entity with favorable therapeutic properties.Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed (see, e.g.,Takasaki et al., 1997, Nature Biotech. 15: 1266-1270). Hereinafter,these and related methods are referred to as “protein structuralanalysis.” These analytical methods may also be used to investigate theinteraction between a receptor protein and peptides selected by phagedisplay, which may suggest further modification of the peptides toincrease binding affinity.

Peptide agonists and antagonists of polypeptides of the disclosure maybe covalently linked to a vehicle molecule. The term “vehicle” refers toa molecule that prevents degradation and/or increases half-life, reducestoxicity, reduces immunogenicity, or increases biological activity oruptake of a therapeutic protein. Exemplary vehicles include an Fc domainor a linear polymer (e.g., polyethylene glycol (PEG), polylysine,dextran, etc.); a branched-chain polymer (see, for example, U.S. Pat.No. 4,289,872; U.S. Pat. No. 5,229,490; WO 93/21259); a lipid; acholesterol group (such as a steroid); a carbohydrate or oligosaccharide(e.g., dextran); or any natural or synthetic protein, polypeptide orpeptide that binds to a salvage receptor or protein transductiondomains.

Antibodies that are immunoreactive with the polypeptides of thedisclosure are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). In the disclosure, specifically bindingantibodies are those that will specifically recognize and bind withPresenilin polypeptides of the disclosure, homologues, and variants, butnot with other molecules. In one embodiment, the antibodies are specificfor the polypeptides of the disclosure and do not cross-react with otherpolypeptides. In this manner, the Presenilin polypeptides of thedisclosure, fragments, variants, fusion polypeptides, and the like, asset forth above, can be employed as “immunogens” in producing antibodiesimmunoreactive therewith.

More specifically, the polypeptides, fragment, variants, fusionpolypeptides, and the like contain antigenic determinants or epitopesthat elicit the formation of antibodies. These antigenic determinants orepitopes can be either linear or conformational (discontinuous). Linearepitopes are composed of a single section of amino acids of thepolypeptide, while conformational or discontinuous epitopes are composedof amino acids sections from different regions of the polypeptide chainthat are brought into close proximity upon polypeptide folding (C. A.Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland PublishingInc., 2nd ed. 1996)). Because folded polypeptides have complex surfaces,the number of epitopes available is quite numerous; however, due to theconformation of the polypeptide and steric hinderances, the number ofantibodies that actually bind to the epitopes is less than the number ofavailable epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can beidentified by any of the methods known in the art. Thus, one aspect ofthe disclosure relates to the antigenic epitopes of the polypeptides ofthe disclosure. Such epitopes are useful for raising antibodies, inparticular monoclonal antibodies, as described in more detail below.Additionally, epitopes from the polypeptides of the disclosure can beused as research reagents, in assays, and to purify specific bindingantibodies from substances such as polyclonal sera or supernatants fromcultured hybridomas. Such epitopes or variants thereof can be producedusing techniques well known in the art such as solid-phase synthesis,chemical or enzymatic cleavage of a polypeptide, or using recombinantDNA technology.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the disclosure, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies can be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies. A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler andMilstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030); and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that producemonoclonal antibodies specific for the polypeptides of the disclosureare also contemplated herein. Such hybridomas can be produced andidentified by conventional techniques. The hybridoma producing the mAbof this disclosure may be cultivated in vitro or in vivo. Production ofhigh titers of mAbs in vivo makes this the most common method ofproduction. One method for producing such a hybridoma cell linecomprises immunizing an animal with a polypeptide; harvesting spleencells from the immunized animal; fusing said spleen cells to a myelomacell line, thereby generating hybridoma cells; and identifying ahybridoma cell line that produces a monoclonal antibody that binds thepolypeptide. For the production of antibodies, various host animals maybe immunized by injection with one or more of the following: aPresenilin-1 polypeptide, a fragment of a Presenilin-1 polypeptide, afunctional equivalent of a Presenilin-1 polypeptide, or a mutant form ofa Presenilin-1 polypeptide. Such host animals may include, but are notlimited to rabbits, mice and rats. Various adjuvants may be used toincrease the immunological response, depending on the host species,including, but not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. The monoclonal antibodies can be recovered by conventionaltechniques. Such monoclonal antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

In addition, techniques developed for the production of “chimericantibodies” (Takeda et al., 1985, Nature, 314:452-454) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a porcine mAb and a humanimmunoglobulin constant region. The monoclonal antibodies of thedisclosure also include humanized versions of murine monoclonalantibodies. Such humanized antibodies can be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen-binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment cancomprise the antigen-binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,Can, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein. Preferably, for use in humans, theantibodies are human or humanized; techniques for creating such human orhumanized antibodies are also well known and are commercially availablefrom, for example, Medarex Inc. (Princeton, N.J.) and Abgenix Inc.(Fremont, Calif.).

Antigen-binding antibody fragments that recognize specific epitopes maybe generated by known techniques. For example, such fragments include,but are not limited to: the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the (ab′)2fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al.,1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,Nature 334:544-546) can also be adapted to produce single chainantibodies against Presenilin-1 gene products. Single chain antibodiesare formed by linking the heavy and light chain fragments of the Fvregion via an amino acid bridge, resulting in a single chainpolypeptide. In addition, antibodies to a Presenilin-1 polypeptide can,in turn, be utilized to generate anti-idiotype antibodies that “mimic” aPresenilin-1 or -2 polypeptide and that may bind to a Presenilin-1 or -2polypeptide using techniques well known to those skilled in the art(see, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff,1991, J. Immunol. 147(8):2429-2438).

Screening procedures by which such antibodies can be identified are wellknown, and can involve immunoaffinity chromatography, for example.Antibodies can be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to cell surface Presenilin-1,induce biological effects (e.g., transduction of biological signals)similar to the biological effects induced when a Presenilin-1 or -2binding partner binds to a cell surface Presenilin-1 or -2. Agonisticantibodies can be used to induce Presenilin-1 or -2-mediated activities,such as epithelial barrier formation, stimulatory pathways, orintercellular communication. Those antibodies that can block binding ofthe Presenilin polypeptides of the disclosure to binding partners forPresenilin-1 can be used to inhibit Presenilin-1 or -2-mediatedepithelial barrier formation, intercellular communication, orco-stimulation that results from such binding. Such blocking antibodiescan be identified using any suitable assay procedure, such as by testingantibodies for the ability to inhibit binding of Presenilin-1 or -2 tocertain cells expressing a Presenilin-1 or -2 binding partner.Alternatively, blocking antibodies can be identified in assays for theability to inhibit a biological effect that results from binding of aPresenilin-1 or -2 to target cells, such as epithelial barrierformation, using assays described herein. Such an antibody can beemployed in an in vitro procedure, or administered in vivo to inhibit abiological activity mediated by the entity that generated the antibody.Disorders caused or exacerbated (directly or indirectly) by theinteraction of Presenilin-1 or -2 with cell surface binding partnerreceptor thus can be treated. A therapeutic method involves in vivoadministration of a blocking antibody to a mammal in an amount effectivein inhibiting Presenilin-1 or -2 binding partner-mediated biologicalactivity. Human or humanized antibodies can be used in such therapeuticmethods. In one embodiment, an antigen-binding antibody fragment isemployed. Compositions comprising an antibody that is directed againstPresenilin-1 or -2, and a physiologically acceptable diluent, excipient,or carrier, are provided herein. Suitable components of suchcompositions are as described below for compositions containingPresenilin polypeptides of the disclosure.

Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or a therapeutic agent, attached to the antibody. Examplesof such agents are presented above. The conjugates find use in in vitroor in vivo procedures. The antibodies of the disclosure can also be usedin assays to detect the presence of the polypeptides or fragments of thedisclosure, either in vitro or in vivo. The antibodies also can beemployed in purifying polypeptides or fragments of the disclosure byimmunoaffinity chromatography.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, e.g., inhibitors, agonists, antagonists, and thelike. Any of these examples can be used to fashion drugs which are moreactive or stable forms of a polypeptide or which enhance or interferewith the function of a polypeptide in vivo (Hodgson J., 1991,Biotechnology 9:19-21, incorporated herein by reference). In oneapproach, the three-dimensional structure of a polypeptide of interest,or of a polypeptide-inhibitor complex, is determined by x-raycrystallography, by nuclear magnetic resonance, or by computer homologymodeling or, most typically, by a combination of these approaches. Boththe shape and charges of the polypeptide must be ascertained toelucidate the structure and to determine active site(s) of thepolypeptide. Less often, useful information regarding the structure of apolypeptide may be gained by modeling based on the structure ofhomologous polypeptides. In both cases, relevant structural informationis used to design analogous Presenilin-like molecules, to identifyefficient inhibitors, or to identify small molecules that may bind aPresenilin of the disclosure. Useful examples of rational drug designmay include molecules which have improved activity or stability as shownby Braxton S and Wells J A (1992, Biochemistry 31:7796-7801) or whichact as inhibitors, agonists, or antagonists of native peptides as shownby Athauda S B et al. (1993, J Biochem 113:742-746), incorporated hereinby reference. The use of Presenilin-1 polypeptide structural informationin molecular modeling software systems to assist in inhibitor design andinhibitor-Presenilin-1 polypeptide interaction is also encompassed bythe disclosure. A particular method of the disclosure comprisesanalyzing the three-dimensional structure of Presenilin polypeptides ofthe disclosure for likely binding sites of substrates, synthesizing anew molecule that incorporates a predictive reactive site, and assayingthe new molecule as described further herein.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described further herein, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypasspolypeptide crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

The polypeptides and peptides of the disclosure also find use ascarriers for delivering agents attached thereto to cells bearingidentified binding partners. The polypeptides thus can be used todeliver diagnostic or therapeutic agents to such cells (or to other celltypes found to express binding partners on the cell surface) in in vitroor in vivo procedures. Detectable (diagnostic) and therapeutic agentsthat can be attached to a polypeptide include, but are not limited to,toxins, other cytotoxic agents, drugs, radionuclides, chromophores,enzymes that catalyze a colorimetric or fluorometric reaction, and thelike, with the particular agent being chosen according to the intendedapplication. Among the toxins are ricin, abrin, diphtheria toxin,Pseudomonas aeruginosa exotoxin A, ribosomal inactivating polypeptides,mycotoxins such as trichothecenes, and derivatives and fragments (e.g.,single chains) thereof. Radionuclides suitable for diagnostic useinclude, but are not limited to, ¹²³I ¹³¹I, ⁹⁹mTc, ¹¹¹In, and ⁷⁶Br.Examples of radionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At,⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu. Such agents canbe attached to the polypeptide by any suitable conventional procedure.The polypeptide comprises functional groups on amino acid side chainsthat can be reacted with functional groups on a desired agent to formcovalent bonds, for example. Alternatively, the polypeptide or agent canbe derivatized to generate or attach a desired reactive functionalgroup. The derivatization can involve attachment of one of thebifunctional coupling reagents available for attaching various moleculesto polypeptides (Pierce Chemical Company, Rockford, Ill.). A number oftechniques for radiolabeling polypeptides are known. Radionuclide metalscan be attached to polypeptides by using a suitable bifunctionalchelating agent, for example. Conjugates comprising polypeptides and asuitable diagnostic or therapeutic agent (preferably covalently linked)are thus prepared. The conjugates are administered or otherwise employedin an amount appropriate for the particular application.

Agents identified by methods provided herein and the peptides of thedisclosure may be administered therapeutically or prophylactically totreat diseases associated with amyloid fibril formation, aggregation ordeposition, regardless of the clinical setting. The compounds of thedisclosure may act to modulate the course of an amyloid related diseaseusing any of the following mechanisms, such as, for example but notlimited to: slowing the rate of amyloid fibril formation or deposition;lessening the degree of amyloid deposition; inhibiting, reducing, orpreventing amyloid fibril formation; inhibiting amyloid inducedinflammation; enhancing the clearance of amyloid from, for example, thebrain; or protecting cells from amyloid induced (oligomers or fibrillar)toxicity.

“Modulation” of amyloid deposition includes both inhibition, as definedabove, and enhancement of amyloid deposition or fibril formation. Theterm “modulating” is intended, therefore, to encompass prevention orstopping of amyloid formation or accumulation, inhibition or slowingdown of further amyloid aggregation in a subject with ongoingamyloidosis, e.g., already having amyloid aggregates, and reducing orreversing of amyloid aggregates in a subject with ongoing amyloidosis;and enhancing amyloid deposition, e.g., increasing the rate or amount ofamyloid deposition in vivo or in vitro. Amyloid-enhancing compounds maybe useful in animal models of amyloidosis, for example, to make possiblethe development of amyloid deposits in animals in a shorter period oftime or to increase amyloid deposits over a selected period of time.Amyloid-enhancing compounds may be useful in screening assays forcompounds which inhibit amyloidosis in vivo, for example, in animalmodels, cellular assays and in vitro assays for amyloidosis. Suchcompounds may be used, for example, to provide faster or more sensitiveassays for compounds. In some cases, amyloid enhancing compounds mayalso be administered for therapeutic purposes, e.g., to enhance thedeposition of amyloid in the lumen rather than the wall of cerebralblood vessels to prevent CAA. Modulation of amyloid aggregation isdetermined relative to an untreated subject or relative to the treatedsubject prior to treatment.

“Inhibition” of amyloid deposition includes preventing or stopping ofamyloid formation, e.g., fibrillogenesis, clearance of soluble Aβ frombrain, inhibiting or slowing down of further amyloid deposition in asubject with amyloidosis, e.g., already having amyloid deposits, andreducing or reversing amyloid fibrillogenesis or deposits in a subjectwith ongoing amyloidosis. Inhibition of amyloid deposition is determinedrelative to an untreated subject, or relative to the treated subjectprior to treatment, or, e.g., determined by clinically measurableimprovement, e.g., or in the case of a subject with brain amyloidosis,e.g., an Alzheimer's or cerebral amyloid angiopathy subject,stabilization of cognitive function or prevention of a further decreasein cognitive function (i.e., preventing, slowing, or stopping diseaseprogression), or improvement of parameters such as the concentration ofAβ or tau in the CSF.

As used herein, “treatment” of a subject includes the application oradministration of a composition comprising an agent identified by amethod of the disclosure to a subject, or application or administrationof a composition of the disclosure to a cell or tissue from a subject,who has a amyloid-β related disease or condition, has a symptom of sucha disease or condition, or is at risk of (or susceptible to) such adisease or condition, with the purpose of curing, healing, alleviating,relieving, altering, remedying, ameliorating, improving, or affectingthe disease or condition, the symptom of the disease or condition, orthe risk of (or susceptibility to) the disease or condition. The term“treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the subject; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating;improving a subject's physical or mental well-being; or, in somesituations, preventing the onset of dementia. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination or apsychiatric evaluation. For example, the methods of the disclosuresuccessfully treat a subject's dementia by slowing the rate of or extentof cognitive decline.

While Alzheimer's disease of the familial or the sporadic type is themajor dementia found in the aging population, other types of dementiaare also found. These include but are not limited to: thefronto-temporal degeneration associated with Pick's disease, vasculardementia, senile dementia of Lewy body type, dementia of Parkinsonismwith frontal atrophy, progressive supranuclear palsy and corticobasaldegeneration and Downs syndrome associated Alzheimers'. Plaque formationis also seen in the spongiform encephalopathies such as CJD, scrapie andBSE. The disclosure is directed to treatment of such neurodegenerativediseases, particularly those involving neurotoxic protein plaques, eg.amyloid plaques.

Downs syndrome is a serious human disorder that occurs with an incidenceof 1 in 800 live births. It is associated with the presence in affectedindividuals of an extra copy of chromosome 21 (trisomy 21). Theβ-amyloid precursor protein (β-APP) gene is encoded on chromosome 21,very close to the Down syndrome locus. All patients with Downs syndrome,if they survive beyond 40 years, develop Alzheimer's-like dementia andthe deposition of Aβ in their brains. There is good reason, therefore,to propose that the over-production of Aβ is connected directly with theoccurrence of the dementia in both AD and Downs syndrome. Therefore, thenature of the identification of therapeutic agents for the ameliorationof the symptoms of AD will also be useful for the amelioration of thesymptoms of Downs syndrome.

“Dementia” refers to a general mental deterioration due to organic orpsychological factors; characterized by disorientation, impaired memory,judgment, and intellect, and a shallow labile affect. Dementia hereinincludes vascular dementia, ischemic vascular dementia (IVD),frontotemporal dementia (FTD), Lewy body dementia, Alzheimer's dementia,etc. The most common form of dementia among older people is Alzheimer'sdisease (AD).

The expressions “mild-moderate” or “early stage” AD are used as synonymsherein to refer to AD which is not advanced and wherein the signs orsymptoms of disease are not severe. Subjects with mild-moderate or earlystage AD can be identified by a skilled neurologist or clinician. In oneembodiment, the subject with mild-moderate AD is identified using theMini-Mental State Examination (MMSE). Herein, “moderate-severe” or “latestage” AD refer to AD which is advanced and the signs or symptoms ofdisease are pronounced. Such subjects can be identified by a skilledneurologist or clinician. Subjects with this form of AD may no longerrespond to therapy with cholinesterase inhibitors, and my have amarkedly reduced acetylcholine level. In one embodiment, the subjectwith moderate-severe AD is identified using the Mini-Mental StateExamination (MMSE). “Familial AD” is an inherited form of AD caused by agenetic defect. A “symptom” of AD or dementia is any morbid phenomenonor departure from the normal in structure, function, or sensation,experienced by the subject and indicative of AD or dementia.

An agent may be administered therapeutically or prophylactically totreat diseases associated with amyloid fibril formation, aggregation ordeposition. The agents of the disclosure may act to, ameliorate thecourse of fibril formation; inhibiting neurodegeneration or cellulartoxicity induced by amyloid-β; inhibiting amyloid-β inducedinflammation; enhancing the clearance of amyloid-β from the brain; orfavoring greater catabolism of Aβ.

An agent may be effective in controlling amyloid-β deposition by actingdirectly on brain Aβ, e.g., by maintaining it in a non-fibrillar form orfavoring its clearance from the brain. The compounds may slow down APPprocessing; may increase degradation of Aβ fibrils by macrophages or byneuronal cells; or may decrease Aβ production by activated microglia.These agents could also prevent Aβ in the brain from interacting withthe cell surface and therefore prevent neurotoxicity, neurodegeneration,or inflammation.

An agent identified by a method provided herein may be used to treatAlzheimer's disease (e.g., sporadic or familial AD). The agent may alsobe used prophylactically or therapeutically to treat other clinicaloccurrences of amyloid-β deposition, such as in Down's syndromeindividuals and in patients with cerebral amyloid angiopathy (“CAA”),hereditary cerebral hemorrhage, or early Alzheimer's disease.

The agent may be used to treat mild cognitive impairment. Mild CognitiveImpairment (“MCI”) is a condition characterized by a state of mild butmeasurable impairment in thinking skills, which is not necessarilyassociated with the presence of dementia. MCI frequently, but notnecessarily, precedes Alzheimer's disease.

Additionally, abnormal accumulation of APP and of amyloid-β protein inmuscle fibers has been implicated in the pathology of sporadic inclusionbody myositis (IBM) (Askanas, V., et al. (1996) Proc. Natl. Acad. Sci.USA 93: 1314-1319; Askanas, V. et al. (1995) Current Opinion inRheumatology 7: 486-496). Accordingly, agents identified by a methodprovided herein may be used prophylactically or therapeutically in thetreatment of disorders in which amyloid-β protein is abnormallydeposited at non-neurological locations, such as treatment of EBM bydelivery of the compounds to muscle fibers.

Additionally, it has been shown that Aβ is associated with abnormalextracellular deposits, known as drusen, that accumulate along the basalsurface of the retinal pigmented epithelium in individuals withage-related macular degeneration (ARMD). ARMD is a cause of irreversiblevision loss in older individuals. It is believed that Aβ depositioncould be an important component of the local inflammatory events thatcontribute to atrophy of the retinal pigmented epithelium, drusenbiogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc. Natl.Acad. Sci. USA 99(18), 11830-5 (2002)).

Accordingly, the disclosure relates generally to methods of treating orpreventing an amyloid-related disease in a subject (preferably a human)comprising administering to the subject a therapeutic amount of an agentor compound identified by a method provided herein, such that amyloidfibril formation or deposition, neurodegeneration, or cellular toxicityis reduced or inhibited. In another embodiment, the disclosure relatesto a method of treating or preventing an amyloid-related disease in asubject (preferably a human) comprising administering to the subject atherapeutic amount of a compound identified by a method describedherein, such that cognitive function is improved or stabilized orfurther deterioration in cognitive function is prevented, slowed, orstopped in patients with brain amyloidosis, e.g., Alzheimer's disease,Down's syndrome or cerebral amyloid angiopathy. These compounds can alsoimprove quality of daily living in these subjects.

Further, the disclosure relates to pharmaceutical compositionscomprising agents for the treatment of an amyloid-related disease, aswell as methods of manufacturing such pharmaceutical compositions.

In general, the agents identified by methods provided herein may beprepared by any method known to the skilled artisan. The agents of thedisclosure may be supplied in a solution with an appropriate solvent orin a solvent-free form (e.g., lyophilized). In another aspect of thedisclosure, the agents and buffers necessary for carrying out themethods of the disclosure may be packaged as a kit. The kit may becommercially used according to the methods described herein and mayinclude instructions for use in a method of the disclosure. Additionalkit components may include acids, bases, buffering agents, inorganicsalts, solvents, antioxidants, preservatives, or metal chelators. Theadditional kit components are present as pure compositions, or asaqueous or organic solutions that incorporate one or more additional kitcomponents. Any or all of the kit components optionally further comprisebuffers.

The therapeutic agent may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

To administer the therapeutic agent by other than parenteraladministration, it may be necessary to coat the agent with, orco-administer the agent with, a material to prevent its inactivation.For example, the therapeutic agent may be administered to a subject inan appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., J. Neuroimmunol. 7, 27(1984)).

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

Suitable pharmaceutically acceptable vehicles include, withoutlimitation, any non-immunogenic pharmaceutical adjuvants suitable fororal, parenteral, nasal, mucosal, transdermal, intravascular (IV),intraarterial (IA), intramuscular (IM), and subcutaneous (SC)administration routes, such as phosphate buffer saline (PBS).

The vehicle can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents are included, for example, sugars, sodiumchloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic agent in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic agent into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and freeze-drying which yields a powder of the activeingredient (i.e., the therapeutic agent) plus any additional desiredingredient from a previously sterile-filtered solution thereof.

The therapeutic agent can be orally administered, for example, with aninert diluent or an assimilable edible carrier. The therapeutic agentand other ingredients may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe subject's diet. For oral therapeutic administration, the therapeuticagent may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic agent in the compositions and preparations may, of course,be varied. The amount of the therapeutic agent in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic agent calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical vehicle. The specification for the dosage unit forms ofthe disclosure are dictated by and directly dependent on (a) the uniquecharacteristics of the therapeutic agent and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such a therapeutic agent for the treatment of amyloiddeposition in subjects.

The disclosure therefore includes pharmaceutical formulations comprisingagents identified by methods described herein, includingpharmaceutically acceptable salts thereof, in pharmaceuticallyacceptable vehicles for aerosol, oral and parenteral administration.Also, the disclosure includes such agents, or salts thereof, which havebeen lyophilized and which may be reconstituted to form pharmaceuticallyacceptable formulations for administration, as by intravenous,intramuscular, or subcutaneous injection. Administration may also beintradermal or transdermal.

In accordance with the disclosure, an agent, and pharmaceuticallyacceptable salts thereof, may be administered orally or throughinhalation as a solid, or may be administered intramuscularly orintravenously as a solution, suspension or emulsion. Alternatively, theagents or salts may also be administered by inhalation, intravenously orintramuscularly as a liposomal suspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired agent, or a salt thereof, or aplurality of solid particles of the agent or salt. The desiredformulation may be placed in a small chamber and nebulized. Nebulizationmay be accomplished by compressed air or by ultrasonic energy to form aplurality of liquid droplets or solid particles comprising the agents orsalts. The liquid droplets or solid particles should have a particlesize in the range of about 0.5 to about 5 microns. The solid particlescan be obtained by processing the solid agent, or a salt thereof, in anyappropriate manner known in the art, such as by micronization. The sizeof the solid particles or droplets will be, for example, from about 1 toabout 2 microns. In this respect, commercial nebulizers are available toachieve this purpose.

A pharmaceutical formulation suitable for administration as an aerosolmay be in the form of a liquid, the formulation will comprise awater-soluble agent, or a salt thereof, in a carrier which compriseswater. A surfactant may be present which lowers the surface tension ofthe formulation sufficiently to result in the formation of dropletswithin the desired size range when subjected to nebulization.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically acceptable vehiclessuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, tragacanth, and sodium alginate; typical-wetting agentsinclude lecithin and polysorbate 80; and typical preservatives includemethyl paraben and sodium benzoate. Peroral liquid compositions may alsocontain one or more components such as sweeteners, flavoring agents andcolorants disclosed above.

Pharmaceutical compositions may also be coated by conventional methods,typically with pH or time-dependent coatings, such that the subjectagent is released in the gastrointestinal tract in the vicinity of thedesired topical application, or at various times to extend the desiredaction. Such dosage forms typically include, but are not limited to, oneor more of cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, andshellac.

Other compositions useful for attaining systemic delivery of the subjectagents include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypiopylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

The compositions of this disclosure can also be administered topicallyto a subject, e.g., by the direct laying on or spreading of thecomposition on the epidermal or epithelial tissue of the subject, ortransdermally via a “patch”. Such compositions include, for example,lotions, creams, solutions, gels and solids. These topical compositionsmay comprise an effective amount, usually at least about 0.1%, or evenfrom about 1% to about 5%, of an agent of the disclosure. Suitablecarriers for topical administration typically remain in place on theskin as a continuous film, and resist being removed by perspiration orimmersion in water. Generally, the carrier is organic in nature andcapable of having dispersed or dissolved therein the therapeutic agent.The carrier may include pharmaceutically acceptable emolients,emulsifiers, thickening agents, solvents and the like.

As described above and further in the Examples below, the disclosuresupports that specific adhesion between β-APP-presenting andPS-presenting cells have different physiological consequences, one atranscellular (juxtacrine) signaling process associated with the normalfunction of these proteins, and the other resulting eventually in theproteolysis of β-APP to form Aβ, leading to the pathology of Alzheimer'sdisease.

G-protein coupled receptors (GPCRs) share a common structural motif.Generally, GPCRs have seven sequences of between 22 to 24 hydrophobicamino acids that form seven alpha helices, each of which spans themembrane. The transmembrane helices are joined by strands of amino acidsbetween transmembrane-2 and transmembrane-3, transmembrane-4 andtransmembrane-5, transmembrane-6 and transmembrane-7 on the exterior, or“extracellular” side, of the cell membrane (these are referred to as“extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).The transmembrane helices are also joined by strands of amino acidsbetween transmembrane-1 and transmembrane-2, transmembrane-3 andtransmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when a ligand binds with the receptor (often referred to as“activation” of the receptor), there is a change in the conformation ofthe intracellular region that allows for coupling between theintracellular region and an intracellular “G-protein.” It has beenreported that GPCRs are “promiscuous” with respect to G proteins, i.e.,that a GPCR can interact with more than one G protein. See, Kenakin, T.,43 Life Sciences 1095 (1988). Although other G proteins exist,currently, Gq, Gs, Gi, Gz and Go are G proteins that have beenidentified. Endogenous ligand-activated GPCR coupling with the G-proteinbegins a signaling cascade process (referred to as “signaltransduction”). Under normal conditions, signal transduction ultimatelyresults in cellular activation or cellular inhibition. It is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Receptor activated G proteins are bound to the inside surface of thecell membrane. They consist of the Gα and the tightly associated Gβγsubunits. When a ligand activates the G protein-coupled receptor, itinduces a conformation change in the receptor (a change in shape) thatallows the G protein to now bind to the receptor. The G protein thenreleases its bound GDP from the Gα subunit, and binds a new molecule ofGTP. This exchange triggers the dissociation of the Gα subunit, the Gβγdimer, and the receptor. Both, Gα-GTP and Gβγ, can then activatedifferent signalling cascades (or second messenger pathways) andeffector proteins, while the receptor is able to activate the next Gprotein. The Gα subunit will eventually hydrolyze the attached GTP toGDP by its inherent enzymatic activity, allowing it to reassociate withGβγ and starting a new cycle.

The alpha subunit of the guanine nucleotide-binding protein G_(o) (“o”for other) mediates signal transduction between a variety of receptorsand effectors. Two forms of Go alpha subunit have been isolated frombrain tissue libraries. These two forms, G_(oA) alpha and G_(oB) alpha(also referred to as G_(oA) and G_(oB)), are the products of alternativesplicing. G_(oA) alpha transcripts are present in a variety of tissuesbut are most abundant in brain. The G_(oB) alpha transcript is expressedat highest levels in brain and testis.

GPCRs comprise one of the largest gene families in the human genome, andmediate a huge variety of cellular functions regulated byneurotransmitters, hormones, chemokines, and many other molecules.Timely uncoupling of GPCR signaling is crucial for maintainingappropriateness and integrity of the GPCR-mediated physiologicalfunctions. This uncoupling is primarily mediated by a much smaller genefamily, currently numbering seven members of GPCR kinases (GRKs). Thespecificity for a few GRK members to regulate a huge numbers of GPCRs iscontrolled in an agonist-dependent manner. In another words, GRKspreferentially bind to and phosphorylate agonist-occupied GPCRs touncouple receptor from corresponding G-protein, a process known ashomologous desensitization. Based on structural similarities, sevenknown GRK members are classified into four subfamilies (GRK1, GRK2/3,GRK4/5/6 and GRK7), with GRK2/3 and GRK5/6 having ubiquitousdistributions including brain. Dysregulation of GRK2, probably GRK5 aswell, has been implicated in the pathogenesis of chronic heart failure,myocardial ischemia, and hypertension, and other cardiovasculardisorders, where the GRKs have been extensively studied. Failure todesensitize rhodopsin signaling by GRK1 can lead to photoreceptor celldeath, and is believed to contribute to retinitis pigmentosa. Inaddition, increased GRK2 levels have been associated with opiateaddiction. Aside from these, however, roles of GRKs in many otherpathological conditions potentially associated with GPCR deregulation,such as in AD, remain virtually unexplored.

Due to the membrane location of GPCRs, GRK's retention on the plasmamembrane or in the cytosol physically affects its access and binding toGPCRs. In resting cells, GRK4 subfamily members (including GRK4/5/6) aretightly associated with the plasma membrane, while GRK2 subfamilymembers (GRK2/3) are primarily cytosolic and translocate to the membranewhen cells are stimulated by GPCR agonists. However, in active cells,subcellular localization of GRKs appears to be determined by the contentand capacity of GRK-binding factors in membrane versus cytosol.Phospholipids, particularly phosphatidylinositol-4,5-biphosphate, appearto play a role in GRKs adherence to the membrane and bind GPCRs, whilephosphatidylserine may also enhance GRK2 binding to GPCRs on themembrane. On the other hand, calcium/calmodulin and othercalcium-binding proteins, as well as actin, actinin, and the like maycontribute to sequester GRKs in the cytosol and inhibit binding of GRKsto GPCRs.

In AD brains, significant membrane alterations, aberrantphosphoinositide metabolism, disrupted calcium homeostasis anddisorganized cytoskeleton proteins could all influence the subcellulardistribution of GRKs. In addition, increased β-amyloid, a hydrophobicpeptide central to AD pathogenesis, has been shown to decrease membranephosphatidylinositol-4,5-biphosphate and increase [Ca²⁺]_(i).

The disclosure is based, in part, upon the demonstration thatPresenilins function as a type of G-protein coupled receptors (GPCRs),resulting in secondary messaging and downstream effects. Evidence of a7-TM structure (like that of rhodopsin) for PS-1 and PS-2 has led to theexamination regarding whether PS-1 and PS-2 belong to the G-Proteincoupled receptor superfamily of proteins, which all share essentially asimilar structure. Although PS does not exhibit any substantial aminoacid homologies with any of the approximately 1,000 GPCR's so farexamined, the fact that all of these GPCR's are 7-TM integral proteins,with many showing no sequence homologies with any others, allows for thepossibility that PS molecules are also GPCRs.

GPCR activity of presenilins was identified using a N141I-PS-2 mutation.The mutation, linked with FAD in Volga German families, caused PC-12cell death in a Pertussis toxin (PTx) sensitive manner. Other studiessuggested that within the 39 amino acid residue carboxyl-terminal domainof PS-1 (located in the cytoplasm in almost all topographic models ofPS-1 in the membrane) there exists a specific binding and regulatingdomain for the brain G_(o) protein. This domain of PS-1 that binds G_(o)in vitro also shows some local amino acid sequence homologies with theG-binding domains of two other GPCR proteins, the D2-dopaminergic, andthe 5HT-1B receptors, as well as the G-protein activating oligopeptide,mastoparan. The possibility that PS-1 as a functional GPCR is furtherdescribed herein.

The disclosure demonstrates that G-protein G_(o) binds full-length PS-1,and is inhibited by Pertussis toxin (PTx). In addition, only G_(oA)binds PS-1, not G_(oB). Transfection of ES null cells with a tail-lessconstruct of PS-1, demonstrates that most of the binding occurs at thecarboxyl terminal tail of PS-1. However, these results indicate thatother cytoplasmic loop regions may be involved in the binding, sincevery small amounts of binding occurred in the presence of tail-lessPS-1. The disclosure also demonstrates that the G-protein binds not onlyto PS-1 but also PS-2 and that for PS-2, in addition to the binding ofG_(oA), G_(oB) also binds intact PS-2. This binding is still presentwhen tail-less PS-2 is used in place of full-length PS-2. These resultssuggest that G_(oB) binds PS-2 at a cytoplasmic domain other than theC-tail. A greater than 700% increase in ³⁵S-GTPγS-labeled Gα_(oA) (butnot Gα_(oB)) binding to PS-1. For PS-2 there is similarly a greater than700% increase over basal levels of ³⁵S-GTPγS-labeled Gα_(oA) binding aswell as ˜300% increase in ³⁵S-GTPγS-labeled Gα_(oB). Treatment with PTxinhibits the incorporation of 35S-GTPγS to both G_(oA) and G_(oB).

Thus, G_(oA) binds to both PS-1 and PS-2 at similar rates, whereas thebinding of G_(oB) to PS-2 is less than half that observed for G_(oA)under the same experimental conditions. The data confirm a functionalconsequence of the G-protein coupling to PS-1 and PS-2 and furthercharacterize the two presenilin proteins as G-protein coupled receptors(GPCRs).

PS-1 and PS-2 appear to have more features in common with family “3”GPCRs (as described above) than with either of the other twofamilies—both have large extra-cellular domains (the N-terminal, and thehydrophilic loop between TM VI and VII), a feature of family 3 GPCRs.Ligand binding in family 3 GPCRs appears to take place exclusively viathe extra-cellular domains, generally the amino terminal domain. TheN-terminal domain of PS-1 or PS-2 is sufficient for in vitro binding ofPS-1 or PS-2 respectively, to β-APP, a proposed ligand and possibleagonist of presenilin GPCR activation. Some family 3 members formhomodimers, usually by di-sulfide bonds via extra-cellular Cys residues.It is well known that PS-1 and PS-2 exist in the membrane as dimers.Further, they both have Cys residues in their extra-cellular domains(7-TM structure. Family 3 GPCRs all have the 3rd intracellular loop asthe shortest loop and this is conserved among each type. Likewise, thethird intracellular loop in PS-1 and PS-2 is the shortest loop,consisting of the sequence KYLPEW (SEQ ID NO:2 from amino acid 239 to243 and SEQ ID NO:4 from amino acid 245 to 250), which is completelyconserved. Some members of family 3 GPCRs interact directly via theircarboxyl terminal PDZ binding domains with intracellular PDZ-domainproteins such as Homer. There is a PDZ binding domain in the carboxylterminal tail of PS-1 which has been shown to bind to several PDZproteins.

PS are expressible at the cell-surface and have 7-TM structures and PS-1and PS-2 participate in a specific cell-cell interaction with β-APP;this β-APP:PS mediated intercellular interaction results in transientincrease in tyrosine kinase activity and protein tyrosinephosphorylation. Furthermore, a β-APP:PS mediated cell-cell interactionis required for at least the major part of the production of Aβ. Theintercellular interaction between β-APP and PS activate G-proteinbinding to PS, due to cross-talk between protein tyrosine kinases andthe G-protein signaling pathways.

Because the disclosure demonstrates G_(o) activation by PS ultimatelyaffects Aβ production, the disclosure provides, in one aspect, a drugtherapy for AD using appropriately designed inhibitors of PS-G_(o)specific binding.

As described herein, experiments were performed to detect such possibleintercellular protein tyrosine phosphorylation signaling events. It wasshown that when cultured DAMI (human megakaryoblast) cells that weretransiently transfected with β-APP were mixed with DAMI cellstransfected with PS-1, or PS-2, within several minutes after mixing, thecell extracts showed significant transient increases in protein tyrosinekinase activity and in phosphotyrosine (PTyr) modification of proteinsubstrates, that did not appear in controls, or in cell mixturescontaining inhibitors of the specific β-APP:PS binding. The downstreamconsequences of this signaling were different depending on whether PS-1or PS-2 was engaged in the intercellular binding to β-APP, because thespectrum of proteins that showed enhanced tyrosine phosphorylation wasaltogether different in the two cases, suggesting a distinction between,rather than a redundancy of, the biochemical functions of the twoclosely homologous PS proteins.

Furthermore, the disclosure demonstrates the biological pathwaysdescribed above by using embryonic stem (ES) cells derived fromPS-1^(−/−), PS-2^(−/−) double null mice herein referred to as ESdouble-null cells, either untransfected in control experiments, ortransfected with β-APP. In the latter case, the β-APP-transfected EScells are mixed with either PS-1- or PS-2-transfected DAMI cells; theDAMI cells do not express significant amounts of endogenous β-APP ontheir surfaces. In this mixed cell-culture system, therefore, theβ-APP-transfected ES double-null cells serve as the only source ofcell-surface expressed β-APP, while the PS-transfected DAMI cells arethe only source of cell-surface expressed PS. If a β-APP:PS specificsignaling event occurs in this system, it is the result of a juxtacrineinteraction between the two cell types. The disclosure demonstrates justsuch an interaction.

Evidence is provided that signaling is accompanied by transientelevations in Src family tyrosine kinase activity, and has identifiedthe individual Src family member mediating the intercellular signalingbetween β-APP and PS-1 to be pp60c-src. In contrast, the β-APP:PS-2signaling involves the Src family member Lyn. These signaling eventsaffect normal physiology. For example, they may play a role in thephysiological defects encountered in the development of β-APP null mice.The Src family of kinases are implicated in cancer, immune systemdysfunction and bone remodeling diseases. For general reviews, seeThomas and Brugge, Annu. Rev. Cell Dev. Biol. 1997, 13, 513; Lawrenceand Niu, Pharmacol. Ther. 1998, 77, 81; Tatosyan and Mizenina,Biochemistry (Moscow) 2000, 65, 49-58; Boschelli et al., Drugs of theFuture 2000, 25(7), 717.

Members of the Src family include the following eight kinases inmammals: Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and Blk. These arenonreceptor protein kinases that range in molecular mass from 52 to 62kD. All are characterized by a common structural organization that iscomprised of six distinct functional domains: Src homology domain 4(SH4), a unique domain, SH3 domain, SH2 domain, a catalytic domain(SH1), and a C-terminal regulatory region. Tatosyan et al. Biochemistry(Moscow) 2000, 65, 49-58. Based on published studies, Src kinases areconsidered as potential therapeutic targets for various human diseases.

GSK-3 activity is also associated with Alzheimer's disease. This diseaseis characterized by the presence of the well-known β-amyloid peptide andthe formation of intracellular neurofibrillary tangles. Theneurofibrillary tangles contain hyperphosphorylated Tau protein, inwhich Tau is phosphorylated on abnormal sites. GSK-3 has been shown tophosphorylate these abnormal sites in cell and animal models.Furthermore, inhibition of GSK-3 has been shown to preventhyperphosphorylation of Tau in cells. In transgenic mice overexpressingGSK3, significant increased Tau hyperphosphorylation and abnormalmorphology of neurons were observed. Active GSK3 accumulates incytoplasm of pretangled neurons, which can lead to neurofibrillarytangles in brains of patients with AD. Inhibition of GSK-3 slows orhalts the generation of neurofibrillary tangles and thus treats orreduces the severity of Alzheimer's disease. Evidence for the role GSK-3plays in Alzheimer's disease has been shown in vitro (see, e.g., Aplinet al. (1996), J Neurochem 67:699; Sun et al. (2002), Neurosci Lett321:61; Takashima et al. (1998), PNAS 95:9637; Kirschenbaum et al.(2001), J Biol Chem 276:7366; Takashima et al. (1998), Neurosci Res31:317; Takashima et al. (1993), PNAS 90:7789; Suhara et al. (2003),Neurobiol Aging. 24:437; De Ferrari et al. (2003) Mol Psychiatry 8:195;and Pigino et al., J Neurosci, 23:4499, 2003). Evidence for the roleGSK-3 plays in Alzheimer's disease has been shown in vivo (See, e.g.,Yamaguchi et al. (1996), Acta Neuropathol 92:232; Pei et al. (1999), JNeuropath Exp Neurol 58:1010; Hernandez et al. (2002), J Neurochem83:1529; De Ferrari et al. (2003) Mol Psychiatry 8:195; McLaurin et al.,Nature Med, 8:1263, 2002; and Phiel et al. (2003) Nature 423:435.

Presenilin-1 and kinesin-1 are also substrates for GSK-3 and relate toanother mechanism for the role GSK-3 plays in Alzheimer's disease, aswas recently described by Pigino, G., et al., Journal of Neuroscience(23:4499, 2003). It was found that GSK3beta phosphorylates kinsesin-1light chain, which results in a release of kinesin-1 from membrane-boundorganelles, leading to a reduction in fast anterograde axonal transport.A mutation in PS-1 may deregulate and increase GSK-3 activity, which inturn, impairs axonal transport in neurons. The consequent reductions inaxonal transport in affected neurons ultimately lead toneurodegeneration.

The disclosure supports that specific adhesion between β-APP-presentingand PS-presenting cells have different physiological consequences, one atranscellular (juxtacrine) signaling process associated with the normalfunction of these proteins, and the other resulting eventually in theproteolysis of β-APP to form Aβ, leading to the pathology of Alzheimer'sdisease. Evidence for a juxtacrine interaction in this system wasobtained with cultured DAMI cells appropriately transfected with eitherβ-APP, or with PS-1 or PS-2; a specific β-APP:PS mediated cell-cellinteraction led to rapid and transient increases in protein tyrosinekinase activity and protein tyrosine phosphorylation within most likelyone, or possibly both, of the adhering cells. DAMI cells were employedbecause these cells do not normally express significant amounts ofendogenous β-APP at the cell surface, and because they are easy todetach mechanically from the cell substratum. Thus, by transfecting ESdouble-null cells with β-APP, cells expressing only surface β-APP butnot PS were made available, and by transfecting DAMI cells with eitherPS-1 or PS-2, additional cells were produced that expressed a PS proteinat the surface, and no significant β-APP.

Mixing experiments between these transfected cells, as the results show,reveal signaling between β-APP and PS (FIG. 5), which result from ajuxtacrine interaction; i.e., a reaction involving membrane-bound PS onone cell surface with β-APP on another. This interaction is specificallyinhibited both by soluble β-APP (the exoplasmic domain of β-APP), and bythe N-terminal domain of PS-1 fused to FLAG, demonstrating the dualspecificity of the interaction of β-APP with PS.

The downstream consequences of this signaling are different depending onwhether PS-1 or PS-2 is engaged in the intercellular binding to β-APP.The spectrum of proteins modified by tyrosine phosphorylation differeddepending on whether PS-1 or PS-2 was involved in the specificintercellular binding to β-APP. The disclosure identifies c-Src as aprotein that undergoes the major transient increases in phosphorylationwhen β-APP and PS-1 interact intercellularly. The Src kinase familymember Lyn appears to be the predominant (or at least a major) Srckinase involved PS-2 intercellular binding to β-APP. Together theseresults show distinct signaling mechanisms that can result in differentrather than redundant physiological functions for the two closelyhomologous presenilin proteins.

The disclosure demonstrates that juxtacrine signaling between β-APP andeither PS-1 or PS-2 results in rapid transient tyrosine kinaseactivation that is different between the two PS proteins. C-Src or Lynare recruited upon the binding of β-APP with PS-1 or PS-2, respectively.Recruitment would suggest that a signaling complex is formed transientlyin vivo at sites of cell-cell contact, setting in motion a cascade ofphosphorylation events that result in developmental consequences.Identifying the region(s) of Src necessary for association with theβ-APP:PS-1 complex provides valuable information regarding the assemblyand activation of a β-APP:PS-1 signaling complex and indicates whetheror not the interaction between the β-APP:PS complex and the kinases isdirect or indirect. β-APP is not known to be phosphorylated oncytoplasmic tyrosine residues, and neither is PS-1, so direct bindingthrough the SH2 domain of c-Src is unlikely since this domain binds onlyat phosphorylated tyrosine residues.

Direct binding can occur via the SH3 domain of Src. SH3 domainsrecognize proline-rich sequences containing the core P-X-X-P (SEQ IDNO:10), where X denotes any amino acid. Ligands recognize the SH3binding surface in one of two opposite orientations. Peptides that bindin a type 1 orientation conform to the consensus sequence R-X-L-P-X-Z-P(SEQ ID NO:11) where Z is normally a hydrophobic or Arg residue (Kay etal., 2000). Interestingly, both PS-1 and PS-2 have a conserved type 1SH3 binding site (LPALP; see SEQ ID NO:2 from amino acid 432 to 436 orSEQ ID NO:4 from amino acid 413 to 417)) in the cytoplasmic carboxylterminal region.

A number of agents that inhibit GPCR interactions are known in the art.In addition, a number of kinase (e.g., c-src, fln and the like)inhibitors are known in the art and can be used in the methods of thedisclosure. Compositions comprising such agents in pharmaceuticallyacceptable carriers for treating AD or which can be used to modulatememory function are contemplated by the disclosure.

Specific GPCR screening assay techniques are known to the skilledartisan. For example, once candidate compounds are identified using the“generic” G protein-coupled receptor assay (e.g., an assay to selectcompounds that are antagonists, agonists, partial agonists, or inverseagonists), further screening to confirm that the compounds haveinteracted at the receptor site can be performed. For example, acompound identified by the “generic” assay may not bind to the receptor,but may instead merely “uncouple” the G protein from the intracellulardomain.

G-protein activity can be determined by assaying enzymes associated witha G-protein. For example, G_(s) stimulates the enzyme adenylyl cyclase.G_(i) (and G_(z) and G_(o)), on the other hand, inhibit this enzyme.Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus,constitutively activated GPCRs that couple the G_(s) protein areassociated with increased cellular levels of cAMP. On the other hand,constitutively activated GPCRs that couple G_(i) (or G_(z), G_(o))protein are associated with decreased cellular levels of cAMP. See,generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, FromNeuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates,Inc. (1992). Thus, assays that detect cAMP can be utilized to determineif a candidate compound is, e.g., an inverse agonist or antagonist tothe receptor (i.e., such a compound would decrease the levels of cAMP).A variety of approaches known in the art for measuring cAMP can beutilized; a most preferred approach relies upon the use of anti-cAMPantibodies in an ELISA-based format. Another type of assay that can beutilized is a whole cell second messenger reporter system assay.Promoters on genes drive the expression of the proteins that aparticular gene encodes. Cyclic AMP drives gene expression by promotingthe binding of a cAMP-responsive DNA binding protein or transcriptionfactor (CREB) that then binds to the promoter at specific sites calledcAMP response elements and drives the expression of the gene. Reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Thus, a constitutively activated Gs-linked receptorcauses the accumulation of cAMP that then activates the gene andexpression of the reporter protein. The reporter protein such asgalactosidase or luciferase can then be detected using standardbiochemical assays.

G_(q) and G_(o) are associated with activation of the enzymephospholipase C, which in turn hydrolyzes the phospholipid PIP₂,releasing two intracellular messengers: diacycloglycerol (DAG) andinisitol 1,4,5-triphosphate (IP₃). Increased accumulation of IP₃ isassociated with activation of G_(q)- and G_(o)-associated receptors.See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8,From Neuron To Brain (3^(rd) Ed.) Nichols, J. G. et al eds. SinauerAssociates, Inc. (1992). Assays that detect IP₃ accumulation can beutilized to determine if a candidate compound is, e.g., an inverseagonist to a G_(q)- or G_(o)-associated receptor (i.e., such a compoundwould decrease the levels of IP₃). G_(q)-associated receptors can alsobeen examined using an Aβ1 reporter assay in that G_(q)-dependentphospholipase C causes activation of genes containing Aβ1 elements;thus, activated G_(q)-associated receptors will evidence an increase inthe expression of such genes, whereby inverse agonists/antagoniststhereto will evidence a decrease in such expression, and agonists willevidence an increase in such expression. Commercially available assaysfor such detection are available.

Similarly, agents or test compound or drug candidates that interact withan extracellular domain or PS-1, PS-2 and/or β-APP and prevent theirnatural intercellular interaction can be used to treat Alzheimer'sDisease and/or reduce Aβ production. Both G-protein activation and Aβproduction are inhibited by specific inhibition of β-APP:PSintercellular interaction. G-protein activation and Aβ production areinhibited by the presence in the co-culture of Pertussis toxin, aninhibitor of G_(o) activation. Demonstrating that the G-proteinactivation that follows β-APP:PS-1 intercellular interaction is on thepathway of Aβ production from β-APP. These results support a direct roleof PS in G-protein signaling and may provide new avenues for thedevelopment of drug candidates for AD.

As described herein a β-APP binding domain of Presenilin refers toeither naturally occurring or synthetic, e.g., protein, oligopeptide(e.g., from about 5 to about 100 amino acids in length, typically fromabout 5 to 50, 5-20 or 5-15 amino acids in length) that binds to a β-APPextracellular domain.

An “agonist” refers to an agent that binds to a polypeptide orpolynucleotide of the disclosure, stimulates, increases, activates,facilitates, enhances activation, sensitizes or up regulates theactivity or expression of a polypeptide or polynucleotide of thedisclosure.

An “antagonist” refers to an agent that inhibits expression of apolypeptide or polynucleotide of the disclosure or binds to, partiallyor totally blocks stimulation, decreases, prevents, delays activation,inactivates, desensitizes, or down regulates the activity of apolypeptide or polynucleotide of the disclosure.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 Daltons and less than about 2500 Daltons, preferably lessthan about 2000 Daltons, preferably between about 100 to about 1000Daltons, more preferably between about 200 to about 500 Daltons.

“Determining the functional effect” refers to assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of presenilin, e.g., measuring physical and chemicalor phenotypic effects of e.g., presenilin interactions with a G-proteinor β-APP. Such functional effects can be measured by any means known tothose skilled in the art, e.g., changes in spectroscopic (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the protein; measuringinducible markers or transcriptional activation of the protein;measuring binding activity or binding assays, e.g. binding toantibodies; measuring changes in ligand binding affinity; measurement ofcalcium influx; measurement of the accumulation of an enzymatic productof a polypeptide of the disclosure or depletion of an substrate; changesin enzymatic activity, measurement of changes in protein levels of apolypeptide of the disclosure; measurement of RNA stability; G-proteinbinding; GPCR phosphorylation or dephosphorylation; tau phosphorylationor dephosphorylation, signal transduction, e.g., receptor-ligandinteractions, second messenger concentrations (e.g., cAMP, IP₃, orintracellular Ca²⁺); identification of downstream or reporter geneexpression (CAT, luciferase, beta-gal, GFP and the like), e.g., viachemiluminescence, fluorescence, colorimetric reactions, antibodybinding, inducible markers, and ligand binding assays. In addition,β-APP binding to presenilin and Aβ production can also be used asdeterminants of a functional effect on presenilin activity.

The working examples provided below are to illustrate, not limit, thedisclosure. Various parameters of the scientific methods employed inthese examples are described in detail below and provide guidance forpracticing the disclosure in general.

EXAMPLES Example 1

cDNAs for G-proteins Gα_(oA) and Gα_(oB) in pcDNA3 were purchased fromUMR cDNA Resource Center, Rolla, Mo. Full-length human PS-1 and PS-2cDNAs in pcDNA3 were cloned by PCR as already described. Tail-lessconstructs of PS-1 and PS-2 were constructed in pcDNA3 in which only thecytoplasmic domain of PS-1 or PS-2 immediately following the lastTM-domain is deleted (this construct comprises of amino acids 1-430 ofPS-1 and 1-410 of PS-2).

Cell culture: ES (PS-1^(−/−)/PS-2^(−/−)) cells were cultured accordingto published protocols.

Transfections: ES (PS-1^(−/−)/PS-2^(−/−)) were transiently transfectedwith 15 μg of pcDNA constructs of full-length human PS-1 or PS-2 and thecDNA of the desired G-proteins using the lipofectamine (Invitrogen)method. Briefly, the lipofectamine-DNA solution was be left at roomtemperature for 30 mins, mixed with enough serum-free medium and addedto the cells. Cells were incubated for 5 h at 37° C. in a CO₂ incubatorafter which the medium was replenished with serum and cells harvested12-24 hours after transfection.

Immunoprecipitations: 24 h after transfection, the culture medium wasremoved, and cells scraped in 200 μl of extraction buffer. Wholecell-extracts were made by sonication, using the solubilizationconditions of Smine et al (50 mM HEPES/NaOH, pH 7.4, 1 mM EDTA, 1 mMDTT, 1% Triton X-100, 60 mM octylglycoside and protease inhibitors). 100μg of each extract was immunoprecipitated using monoclonal antibodies tothe large loop of PS-1 (MAB5232) or PS-2 (MA1-754). Theimmunoprecipitated proteins were next separated on 12% SDS PAGE andtransferred to a membrane. Western blot hybridization against antibodiesto the G protein G_(o) (K-20, sc-387 from Santa Cruz Biotechnology,affinity purified; this polyclonal antibody recognizes both G_(oA) andG_(oB)) was then carried out.

Western blot hybridizations: Immunoprecipitated proteins were boiled for5 min in loading buffer (50 mM Tris, pH 6.8, 0.1 M DTT, 2% SDS, 0.1%bromophenol blue, 10% glycerol), separated electrophoretically onSDS-PAGE (12%) gels, and the proteins transferred onto nitrocellulosefilters. Filters were incubated with the primary polyclonal rabbitG-protein antibodies followed by horse radish peroxidase-conjugated goatanti-rabbit IgG. Filter-bound peroxidase activity was detected bychemiluminescence.

Binding of G-protein G_(o) to PS-1 ES (PS-1^(−/−)/PS-2^(−/−)) cells weretransiently transfected with cDNA to full-length human PS-1 and the cDNAof the G-proteins G_(oA) or G_(oB) (UMR cDNA Resource Center, Rolla,Mo.). 24 h after transfection, whole cell-extracts were made bysonication, using the solubilization conditions of Smine et al (50 mMHEPES/NaOH, pH 7.4, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 60 mMoctylglycoside and protease inhibitors). 100 μg of each extract wasimmunoprecipitated using monoclonal antibodies to the large loop, whichis extracellular in the 7-TM model (Mab #5232, Chemicon, which was usedin previous published work). The immunoprecipitated proteins were nextseparated on 12% SDS PAGE and transferred to a membrane. Western blothybridization against antibodies to both, PS-1 and G_(o) (K-20, sc-387from Santa Cruz Biotechnology, affinity purified; this polyclonalantibody recognizes both G_(oA) and G_(oB)) was then carried out.

Binding of G-protein G_(o) to PS-2: ES (PS-1^(−/−)/PS-2^(−/−)) weretransiently transfected with cDNA of full-length human PS-2 and the cDNAof the G-proteins G_(oA) or G_(oB) (UMR cDNA Resource Center, Rolla,Mo.). 24 h after transfection, whole cell-extracts were made bysonication, using the solubilization conditions of Smine et al (50 mMHEPES/NaOH, pH 7.4, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 60 mMoctylglycoside and protease inhibitors). 100 μg of each extract wasimmunoprecipitated using mouse monoclonal antibodies to the large loopof PS-2 (MA1-754 from Affinity BioReagents). The immunoprecipitatedproteins were next separated on 12% SDS PAGE and transferred to amembrane. Western blot hybridization against antibodies to both, PS-2and G_(o) was then carried out.

Pertussis Toxin Treatment: The PTx protomer was incubated with 10 mM DTTat 37° C. for 10 min to convert it to its enzymatically active form. 5 hafter transfecting ES cells with PS-1 or PS-2 and the G-protein cDNAs,500 ng/ml of activated PTx was added to the cells in culture medium inthe presence of 1 mM NAD, 2 mM MgCl₂ and 1 mM EDTA and the cellsincubated at 37° C. in the presence of 5% CO₂ for 12 h. Cells were thenharvested and examined for [³⁵S]GTPγS incorporation as described below.

GTPγS Binding: Cells were harvested and proteins solubilized bysonication in solublilization buffer (50 mM HEPES/NaOH pH 7.4, 1 mMEDTA, 1 mM DTT, 1% Triton-X100, 60 mM octylglycoside, 1× Proteaseinhibitor mix). 100 μg of protein was mixed with an equal volume ofBuffer B (50 mM HEPES/NaOH pH 7.4, 40 μM GDP, 50 mM MgCl₂, 100 mM NaCl)in a volume of 200 μl. The reaction was started with 50 nM [³⁵S]GTPγS(1250 Ci/mmol) and incubation carried out for 60 min at RT after whichthe reaction was stopped by the addition of 20 μl of 10× Stopping buffer(100 mM Tris-Hcl, pH 8, 25 mM MgCl₂, 100 mM NaCl, 20 mM GTP. The samplewas then immunoprecipitated with anti-PS-1 loop monoclonal antibody (5μl). The antibody-protein complex was subjected to binding to ProteinA/G agarose for 90 min at RT and washed twice with washing buffer 1 (50mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 1% Triton X-100 1× proteaseinhibitor mix, 150 mM NaCl and 60 mM octyl-β-D-glucopyranoside), andonce with each of washing buffers 2 (50 mM HEPES, pH 7.4, 1 mM EDTA, pH8.0, 0.5% Triton X-100, 1× protease inhibitor mix and 50 mM NaCl) and 3(50 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0 and 1× protease inhibitor mix.The washed agarose beads were then suspended in scintillation fluid(CytoScint, ICN) (5 ml) and counted in a Beckman Coulter LS 6000 SCscintillation counter for 3 min.

When 100 μg of extract of ES (PS-1^(−/−)/PS-2^(−/−)) cellsco-transfected with cDNAs for full-length human PS-1 and the G-proteinGα_(oA) or Gα_(oB) were immunoprecipitated with MAb to the largehydrophilic loop of PS-1, followed by Western blot hybridization withaffinity purified polyclonal antibody to G_(o) (which recognizes bothisoforms, G_(oA) and G_(oB)), only the PS-1/G_(oA) co-transfected cellsgave a robust signal for G_(o) at ˜45 kDa (FIG. 1, lane 3), suggestingthat G_(oA), but not G_(oB), binds to PS-1. Control untransfected cellsor cells transfected with PS-1 alone did not show a G_(o) band onWestern blots when treated identically (FIG. 1).

Verification of the binding of G-protein G_(o) to the cytoplasmiccarboxyl terminus of PS-1. A tail-less construct of PS-1 was made inpcDNA3 in which only the cytoplasmic domain of PS-1 immediatelyfollowing the last TM-domain is deleted (this construct comprises aminoacids 1-430). This construct was used to transfect ES(PS-1^(−/−)/PS-2^(−/−)) cells. Tail-less PS-1 has been shown tointegrate into the membrane and to be expressed at the cell surface. Inan identical strategy to the one described above for full-length PS-1,ES (PS-1^(−/−)/PS-2^(−/−)) cells were transfected with cDNAs fortail-less PS-1 and the G-proteins G_(oA) or G_(oB). Cells extracts werethen subjected to immunoprecipitation with PS-1 loop MAb #5232),separated on SDS PAGE and Western blotted with antibodies to G_(o).

100 μg of extract of ES (PS-1^(−/−)/PS-2^(−/−)) cells co-transfectedwith cDNAs for tail-less PS-1 and the G-protein Gα_(oA) or Gα_(oB) wereimmunoprecipitated with MAb to the large hydrophilic loop of PS-1,followed by Western blot hybridization with affinity purified polyclonalantibody to G_(o) (recognizes both isoforms, G_(oA) and G_(oB)). Bindingwas detected (FIG. 1, lane 6) indicating that the carboxyl terminal 39amino acids earlier identified to be the binding domain did notconstitute the entire binding domain of PS-1 for G_(oA). G_(oB) showedno binding to tail-less PS-1 (FIG. 1, lane 7).

The results using the tail-less construct, which eliminated the majorpart of G_(oA) binding to PS-1, show specificity for some PS-1:G_(oA)binding to another region of PS-1 besides the PS-1 tail. They also ruleout the possibility that G_(oA) may have bound to other components ofthe PS-1 β-secretase complex, that may have co-immunoprecipitated withthe PS-1 antibody.

Additional studies were performed to elucidate the binding of G-proteinG_(o) to intact PS-2. The 39 amino acid PS-1 C-terminal regionidentified to be the binding domain is completely conserved in theC-terminal tail of PS-2. Accordingly, it was believed that theC-terminal domain of PS-2 would also bind Gα_(o). As with PS-1, G_(o)was shown to bind to PS-2, but with distinct differences. The G_(o)antibody, which recognizes both G_(oA) and G_(oB), showed a doublet onWestern blots of PS-2 immunoprecipitates of extracts of cellsco-transfected with PS-2 and G_(oA) as well as PS-2 and G_(oB) cDNAs.The doublet presumably represents binding of both isoforms of G_(o) toPS-2 (FIG. 3, lanes 2 and 4). In contrast, PS-1 did not bind to G_(oB)(FIG. 1, lane 4) and only showed a single band on Western blots with thesame G_(o) antibody (FIG. 1, lane 3).

The binding of G-protein G_(o) to the cytoplasmic carboxyl terminus ofPS-2 was investigated. As for PS-1, a tail-less construct of PS-2 wasmade in pcDNA3 in which only the cytoplasmic domain of PS-2 immediatelyfollowing the last TM-domain was deleted (this construct comprised aminoacids amino acids 1-410). This construct was used to transfect ES(PS-1^(−/−)/PS-2^(−/−)) cells and has been shown to integrate into themembrane and be expressed at the cell surface (FIG. 2). In an identicalstrategy to the one described above for full-length PS-1 and PS-2, ES(PS-1^(−/−)/PS-2^(−/−)) cells were transfected with cDNAs for tail-lessPS-2 and the G-proteins G_(oA) and G_(oB). Cells extracts were thensubjected to immunoprecipitation with PS-2 loop Mab # MA1-754),separated on SDS PAGE and Western blotted with antibodies to G_(o).

When tail-less PS-2, co-expressed with G_(oA) was immunoprecipitatedwith PS-2 MAb and Western blotted with anti G_(o) antibody, as withresults for PS-1, there was a decrease in band intensity, but the bandwas not totally absent. The intensity of the bands in the G_(oB)/PS-2co-transfection sample, on the other hand, was unaltered for thetail-less sample suggesting that G_(oB) binds PS-2 at an intracellulardomain other than the carboxyl terminal tail FIG. 3, lanes 3 and 5).Therefore, PS-1 and PS-2 are discriminated not only by the G_(o)isoforms that they bind to, but also the binding sites on the PS-1 andPS-2 that are not homologous to one another. It seems likely, therefore,that functional studies of PS-1 and PS-2 will give quite differentresults; i.e., PS-1 and PS-2 are not merely functionally redundantproteins.

Additional studies of PS mediated functional activation of Gα_(oA) andGα_(oB) PS-1 and the G-proteins G_(oA) and G_(oB) were performed.Previous studies used GTP hydrolysis and GTPγS binding as one of severalindependent approaches to evaluate G_(o) binding to the carboxylterminus of PS-1. However, they carried out this assay with asynthesized peptide of residues 429-467 in the C-terminus of PS-1, alongwith three control peptides. The approach on the other hand was toevaluate the functional consequences of the binding of the G-proteinsG_(oA) and G_(oB) to intact PS-1 and PS-2 in the co-transfected cell, byassaying for 35S-GTPγS incorporation in cell extracts.

The ³⁵S-GTPγS incorporation in extracts of ES cells that wereco-transfected with cDNAs for PS-1 and the G-protein G_(oA) was shown tobe over 700% the value obtained for control untransfected ES (PS^(−/−))cells (FIG. 4, lane 2). This increase was not seen when cellstransfected with PS-1 and G_(oA) cDNAs were first treated with PTx (FIG.4, lane 3) showing an inhibition of function in the presence of thetoxin. Cells transfected with cDNAs for PS-1 and G_(oB) on the otherhand did not show incorporation of ³⁵S-GTPγS (FIG. 4 lane 4), consistentwith previous results of a lack of binding of G_(oB) to PS-1.

As with PS-1, PS-2 when co-expressed with G_(oA) and assayed for³⁵S-GTPγS binding showed greater than 700% increase in ³⁵S-GTPγS bindingover untransfected control ES (PS^(−/−)) extracts (FIG. 4, lane 2). Thiswas inhibited in the presence of PTx (FIG. 4, lane 3). Unlike the casefor PS-1, G_(oB) binding to PS-2 does give an increase in ³⁵S-GTPγSincorporation. This novel finding is consistent with other data providedherein indicating that G_(oB) binds to PS-2 but not PS-1. The increasein ³⁵S-GTPγS incorporation is less than that observed for G_(oA) (˜300%)(FIG. 4, lane 4). This increase is inhibited in the presence of PTx. Theresults shown in FIG. 4 are representative of at least 3 independentexperiments.

Example 2

ES PS double-null cells were cultured and plated overnight. The cellswere transfected with a pcDNA3 construct of full-length human β-APP cDNAusing lipofectamine (Invitrogen) according to the manufacturer'sprotocols. DAMI cells were cultured and transfected either with pcDNA3or with a pcDNA3 construct of full-length human PS-1 or PS-2 cDNA.

Affinity-purified polyclonal rabbit anti-PTyr antibodies (Maher et al.,1985) were used in Western blots. A mouse monoclonal anti-PTyr antibody(4G10; Upstate Biotechnology, Lake Placid, N.Y.) was used in ELISAanalyses. Mouse monoclonal antibody to human pp60c-src (Anti-Src, cloneGD11) and rabbit polyclonal antibody to Lyn (Anti-Lyn) were purchasedfrom Upstate Biotechnology. Rabbit polyclonal antibody to Fyn (Anti-Fyn,sc-16) was purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.Primary rat anti-human PS-1 monoclonal antibody MAb #1563 directed tothe N-terminal domain of PS-1 was purchased from Chemicon International,Temecula, Calif. It was raised to a fusion protein antigen containingpart of the N-terminal domain of human PS-1 (residues 21-80) fused toGST. Primary mouse monoclonal antibody MAb #348 to the human β-APPextracellular domain was purchased from Chemicon International.

Fluorescein isothiocyanate (FITC)-conjugated affinity purified goatanti-rat IgG and tetramethylrhodamine B isothiocyanate(TRITC)-conjugated affinity purified donkey anti-mouse IgG secondaryantibodies were purchased from Jackson ImmunoResearch, West Grove, Pa.Immunofluorescence labeling Transfected and untransfected DAMI cellswere fixed with 4% paraformaldehyde in PBS for 10 mins and used withoutpermeabilization. Cells were labeled in suspension with antisera to PS-1(1:200 dilution), and β-APP (1:500 dilution) in PBS containing 1% BSAfor 30 min at room temperature. After washing with PBS three times bycentrifugation, the cells were resuspended in 1% BSA/PBS and incubatedwith appropriate fluorescent secondary antibodies. Incubation wascarried out at room temperature for 20 min, after which the cells werewashed with PBS and mounted onto slides in the presence of mountingmedium (Vector Laboratories, Burlingame, Calif.).

Immunofluorescent microscopy was performed using oil immersion with aX60 objective lens. The slides were viewed using fluoresceinisothiocyanate and tetramethylrhodamine B isothiocyanate filters and aZeiss Photoscope III instrument, or with Nomarski optics.

N-terminal domains of PS-1 and PS-2 were obtained by PCR and cloned intothe Tth 111 I and Xho-1 sites of the FLAG expression vector (ScientificImaging Systems, IBI 13100) to produce a fusion protein with FLAGattached at the N-terminus of either the PS-1- or 2 N-terminal domains.The two FLAG-fusion proteins were grown separately in DH5α bacteria andaffinity purified according to the manufacturer's protocols. Thepurified recombinant proteins were checked by Western blots usingantibodies to both FLAG and either the N-terminal domain of PS-1 orPS-2.

DAMI:ES cells: Equal numbers (0.5×10⁶/ml) of β-APP 695 (Selkoe andPodlisny, 2002) -transfected ES double-null cells and PS-1 transfectedDAMI cells were co-cultured at 37° C. for various times between 0-20mins.

All experiments after those in FIG. 7 (with the exception of FIG. 9a ,Panel 4) were carried out with appropriately transfected DAMI cellsonly. Equal numbers (0.5×10⁶/ml) of β-APP-transfected DAMI cells andeither PS-1- or PS-2-transfected DAMI cells were mixed gently at roomtemperature, exactly as described (Dewji and Singer, 1998). In controlexperiments, DAMI cells transfected with pcDNA3 alone were substitutedfor the β-APP transfected cells.

At several times between 0 and 20 min after mixing, an aliquot of eachcell mixture was rapidly centrifuged, the culture medium was removed,and the cell pellet was suspended in 200 μl of extraction buffer (50 mMTris, pH 8.0/150 mM NaCl/0.5% Nonidet-P40) containing proteaseinhibitors (1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride(AEBSF)/1 μg/ml antipain/0.1 μg/ml pepstatin A/0.1 μg/ml leupeptin) andthe phosphatase inhibitor sodium orthovanadate (0.1 mM). The mixture wassonicated with three bursts of 20 sec duration and then centrifuged.These extract supernatants were then used for Western blot and ELISAanalyses as described below.

Assays for Src family of protein tyrosine kinases in cell extracts wereperformed. The substrate peptide {[Lys19]cdc2 (6-20)-NH2} and controlpeptides {[Lys19Ser14Val12]cdc2 (6-20)} and {[Lys19Phe15]cdc2(6-20)}were purchased from Upstate Biotechnology Inc. Src kinase activity wasmeasured in extracts of transfected DAMI cells (either β-APP- orpcDNA3-transfected) mixed with PS-1-transfected cells; and with β-APP-or pcDNA3-transfected cells mixed with PS-2-transfected cells, using allthree peptides. Controls included experiments carried out using nosubstrate in the reaction mixture.

The substrate peptide (1.5 mM in 10 μl), Src kinase reaction buffer (100mM Tris-HCl, pH 7.2, 125 mM MgCl₂, 25 mM MnCl₂, 2 mM EGTA, 0.25 mMsodium orthovanadate, 2 mM DTT) (10 μl), Src kinase (2-20 U of purifiedenzyme per assay or 10-200 μg protein lysate in 10 μl and [γ-³²P]ATP(NEN Dupont, Boston, Mass.) diluted with Mn²⁺/ATP cocktail (10 μl), wereincubated for 15-20 min at 30° C.

Aliquots of the extract supernatants described above (100 μgprotein/lane) were boiled for 5 min in loading buffer (50 mM Tris, pH6.8, 0.1 M DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), separatedelectrophoretically on SDS-PAGE (10%) gels, and the proteins transferredonto nitrocellulose filters. Filters were incubated with the primarypolyclonal rabbit anti-PTyr antibodies followed by the horse radishperoxidase-conjugated goat anti-rabbit IgG. Filter-bound peroxidaseactivity was detected by chemiluminescence.

Cell lysates were prepared in extraction buffer and clarified bymicro-centrifugation at 4° C. for 15 mins.

Extracts were incubated with 4 μg antibodies specific for either c-Src,Lyn or Fyn followed by protein-A or G sepharose (40 μl of slurry). Theantigen antibody-protein-A (or -G) sepharose complex was washed threetimes in RIPA (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1%Na deoxycholate, 0.1% SDS, 1% trasylol, 25 μM leupeptin) containing 300mM NaCl, once with RIPA containing 10 mM NaCl, twice with 40 mMTris-HCl, pH 7.2 and once with kinase buffer containing 25 mM HEPES, pH6.9, 3 mM MnCl₂ and 200 μM sodium orthovanadate.

Reactions were performed according to published protocols (Zisch et al.,1998) in 40 μl kinase buffer (25 mM Hepes, pH 6.9, 3 mM MnCl₂ and 200 μMsodium orthovanadate) containing 5 μCi [g32P] ATP (3000 Ci/mmol) for 30min at 37° C. The reaction beads were washed three times with kinasebuffer and resuspended in 75 μl SDS gel loading buffer (250 mM Tris-HCl,pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 0.02% bromophenol blue and 75%glycerol). Autophosphorylation reactions were subjected to SDS-PAGEfollowed by transfer of proteins onto PVDF membranes andautoradiography.

ELISAs Protein tyrosine kinase activity was measured by an Enzyme LinkedImmunosorbent Assay (ELISA) using a tyrosine kinase assay kit (UpstateBiotechnology). A biotinylated substrate peptide containing tandemrepeats of Poly (Glu4-Tyr) was incubated with supernatants of extractsof transfected cells mixed for different times (20 μg protein/well) inthe presence of non-radioactive ATP and a Mn²⁺/Mg²⁺ co-factor cocktailaccording to the manufacturer's protocols. A phosphotyrosine specificmouse monoclonal antibody (4G10) conjugated to horseradish peroxidasewas used to detect the phosphorylated substrate by ELISA.

Absence of cell surface expression of β-APP in untransfected andPS-1-transfected DAMI cells. Because the initial set of studies dependson the proposition that DAMI cells, after transfection with PS-1,continue to express only negligible amounts of β-APP on their surface,the following experiments were first carried out. Both untransfected andPS-1-transfected DAMI cells in the fixed but impermeable state weredoubly immunofluorescently labeled for β-APP and PS-1. Untransfectedfixed impermeable DAMI cells, as previously shown (Querfurth and Selkoe,1994), do not express significant amounts of β-APP at the cell surface(FIG. 6a , Panel 2), whereas DAMI cells transfected with a pcDNA3construct of β-APP show substantial cell-surface expression in fixedimpermeable cells (FIG. 5b , Panel 2). FIGS. 6a and b , Panels 1 show,however, that untransfected fixed impermeable DAMI cells do expressendogenous cell-surface PS-1. In FIG. 5c , Panel 1, this cell-surfaceexpression of PS-1 is increased in fixed impermeable PS-1-transfectedcells. FIG. 5c , Panel 2, shows that transfecting DAMI cells with PS-1does not significantly increase the cell-surface expression of β-APPover the negligible levels seen in untransfected cells (FIG. 5a , Panel2). FIG. 2d , Panel 2, shows cell-surface expression of β-APP in ESdouble-null fixed impermeable cells transfected with β-APP, but not PS-1expression (FIG. 5d , Panel 1).

With untransfected, fixed impermeable ES double-null cells, there is, asexpected, no labeling for cell-surface PS-1 (FIG. 5e , Panel 1), but asmall amount of surface expression of endogenous β-APP (FIG. 5e , Panel2). These results confirm that in interactions of β-APP-transfected ESdouble-null cells and PS-transfected DAMI cells, only the ES cellsexpress cell-surface β-APP, and no PS; while only the PS-transfectedDAMI cells express PS, and no β-APP, at the cell-surface. If a β-APP:PSinteraction occurs after cell mixing, it can therefore only be theresult of a cell-cell interaction.

Also provided herein are data indicating that specific β-APP:PSintercellular signaling results in an increase in tyrosine kinaseactivity. ES double-null cells transfected with β-APP were mixed withDAMI cells transfected with PS-1, and were co-cultured for various timesbetween 0-20 min, using cell densities that ensured cell-cell contact.ELISA assays were then carried out on cell extracts to measure proteintyrosine kinase activity. FIG. 6a shows that these co-cultures produceda rapid and transient increase in protein tyrosine kinase activitysimilar in extent and kinetics to those previously described whenPS-1-transfected DAMI cells were mixed with β-APP-transfected DAMI cells(Dewji and Singer, 1998). When the same interaction as in FIG. 6a wascarried out in the presence of 25 μg of purified baculovirus-derivedsoluble β-APP (extra-cellular domain of β-APP) (FIG. 6b ) or 25 μg offusion peptide of the FLAG reporter fused to the N-terminal domain ofPS-1 (FIG. 6c ), no increase in protein tyrosine kinase activityresulted. On the other hand, the same β-APP:PS-1 co-cultures in thepresence 25 μg of FLAG-PS-2 N-terminal domain fusion peptide did notinhibit PTyr formation (FIG. 2d ). These results clearly establishseveral points: 1) Soluble β-APP itself does not activate thePS-1-transfected DAMI cells to exhibit tyrosine kinase activity; theintact β-APP in the transfected ES cell membrane is required. On thecontrary, the soluble β-APP inhibits the activity produced by themembrane-bound β-APP, demonstrating that membrane-bound β-APP isspecifically involved in the activation; 2) the N-terminal domain ofPS-1 is itself incapable of activating the β-APP-transfected cell toexhibit tyrosine kinase activity. The intact PS-1 molecule in its DAMIcell membrane is required. But the N-terminal domain of PS-1 (but notPS-2) inhibits the activation of the co-culture, showing thatmembrane-bound PS-1 on the PS-1-transfected DAMI cell is alsospecifically involved in the interaction; 3) The protein nature of theinhibitors, soluble β-APP and the FLAG-fusion protein of the N-terminaldomain of PS-1, assures their impenetrability of the cell membranes ofliving DAMI and ES cells, and therefore demonstrates that it is only theexterior domains of the cell-surface β-APP and PS-1 that are involved ingenerating the signaling event (i.e., the signaling is of the juxtacrinetype). These results provide compelling evidence that establish that ajuxtacrine interaction between β-APP and PS can occur.

Furthermore, this demonstration that the N-terminal domain of PS-1 isexposed at the extracellular surface is consistent with the 7-TMtopography of the PS proteins, but is contrary to the prediction of theβ-TM model, which positions the N-terminal domain of PSintra-cellularly.

Additional data provided herein indicate that β-APP:PS-1 and β-APP:PS-2intercellular signaling can be mediated by members of the Src family oftyrosine kinases. The increases in PTyr modification that are aconsequence of β-APP:PS intercellular binding involved one or moreprotein tyrosine kinases that need to be identified. Since neither β-APPnor the PS proteins contain such a kinase active site, an indirectactivity of the cytoplasmic domains of these proteins, such as thedirect or indirect binding of a cytoplasmic tyrosine kinase to one ofthese domains, may be involved in the downstream signal. Since severalcytoplasmic tyrosine kinases have been identified within the Src genefamily, Src family protein tyrosine kinases were assayed in cellextracts of mixed transfected cells using the substrate peptide[lys19]cdc2(6-20)-NH₂ (KVEKIGTYGVVKK; SEQ ID NO:12). This peptide, withTyr 19 in cdc2(6-20) replaced by lys, has been shown to be an efficientsubstrate for the Src family kinases. All Src family kinases tested,including v-Src and c-Src, c-Yes, Lck, Lyn and Fyn, demonstrate strongactivity towards this substrate. Two control peptides were also used: Inthe first peptide, [lys19ser14val12]cdc2(6-20)NH2 (KVEKIGVGSYGVVKK; SEQID NO:13), glu12 and thr14 were replaced by val and ser, respectively,causing a significant decrease in efficiency of the resulting peptide toserve as a substrate for the Src family tyrosine kinases. The otherpeptide, [lys19phe15]cdc2(6-20)NH2 (KVEKIGEGTFGVVKK; SEQ ID NO:14)should not be phosphorylated by tyrosine kinases but did contain apotential target for ser/thr kinases (thr 14).

The results of Src family kinase activity measurements in extracts ofco-cultures of β-APP-transfected DAMI cells with PS-1-transfected DAMIcells evoking β-APP:PS-1 interactions, and for the corresponding controllacking β-APP (pcDNA3:PS-1), are shown in FIGS. 7a and b . Similarresults for transfected DAMI cell mixtures evoking β-APP:PS-2interactions, and extracts of control pcDNA3:PS-2 mixed transfected DAMIcells, using these three peptides are shown in FIGS. 7c and d . For eachβ-APP:PS cell mixture, where [lys19]cdc2(6-20)NH2 was used as the Srcfamily kinase substrate, the temporal course of increased activitycompared to control peptides were obtained that paralleled ELISA resultsfor tyrosine kinase activity. For the β-APP:PS-1 interaction (FIG. 7a ),Src family kinase activity peaked at 8 minutes and returned to baselinelevels by 12 minutes confirming previous ELISA results for tyrosinekinase activity as a function of time after cell mixing. No significantincrease could be observed when the same substrate was used for thecontrol pcDNA3:PS-1 (FIG. 7b ) mixed cells. For the cell mixturesevoking β-APP:PS-2 interactions (FIG. 7c ), as for the tyrosine kinaseELISA results, two clear peaks of activity were observed with substratepeptide [lys19]cdc2(6-20)NH₂, at 9 and 16 minutes after mixing.

For the corresponding control lacking β-APP, pcDNA3:PS-2 (FIG. 7d ), nosignificant increases of Src kinase activity over background wereobserved. These results suggest that the increases in tyrosine kinaseactivity previously observed for β-APP- with PS-1-transfected cellmixtures, or β-APP- with PS-2-transfected cell mixtures, involve one ormore members of the Src tyrosine kinase family.

Inhibition of tyrosine kinase activity in the presence of specificinhibitors of Src family kinases and tyrosine kinase. The involvement ofthe Src kinase family in β-APP:PS intercellular signaling was furtherconfirmed with ELISAs of extracts of β-APP:PS-1 mixed cell interactionscarried out in the presence or absence of specific inhibitors oftyrosine kinase (herbimycin A) and Src family kinases (PP2). FIG. 8ademonstrates that in the presence of 10 μg/ml herbimycin A, the increasein tyrosine kinase activity at β-10 mins after mixing β-APP-transfectedDAMI cells with PS-1-transfected DAMI cells is completely inhibited. Thesame experiment carried out in the presence of 10 nM PP2 (FIG. 8b )similarly showed the inhibition of tyrosine kinase activity.

Additional data related to the involvement of c-Src in β-APP:PS-1intercellular signaling is provided below. In order to determine theidentity of the Src family member(s) involved in the β-APP:PS-1intercellular signaling, we began by investigating pp60c-Src. Two mainprotein bands of apparent molecular weights 58 and 60 kDa, a doubletsimilar in size to c-Src, underwent transient PTyr modification in thisjuxtacrine interaction. When extracts of mixtures of PS-1-transfectedDAMI cells with β-APP-transfected DAMI cells were subjected to SDS16PAGE and immunoblotting with either anti-PTyr or anti-c-Src antibodies,both antibodies reacted with the same two bands (FIG. 9a , Panels 1-3).Panel 1 of this figure immunoblotted with anti-PTyr antibodies showstransient increases in tyrosine phosphorylation of the protein bandswith a maximum at 8-10 mins after cell mixing. In Panel 2 the sameextracts immunoblotted with the c-Src antibody show no variation withtime, indicating that the c-Src protein concentration remains unchangedduring the increase in its PTyr levels. An important observation wasthat when ES double-null cells transfected with β-APP (thereforeexpressing only β-APP, but no PS-1 or 2) were mixed with DAMI cellstransfected with PS-1 (therefore expressing only PS-1, but no cellsurface β-APP), the p60 c-Src proteins plus one or two additionalproteins underwent transient increases in PTyr modification at similartimes after mixing (FIG. 9a , Panel 4) that were seen with theβ-APP-transfected DAMI cells mixed with PS-1-transfected DAMI cells(FIG. 9a , Panel 1). The PTyr modification results were thereforeassociated with PS-1 and not the cell type that expressed it (see belowfor PS-2).

In order to test further whether c-Src was the member of the tyrosinekinase family that underwent transient tyrosine phosphorylation in theβ-APP:PS-1 interaction, experiments were carried out(autophosphorylation) in which the extracts of the mixed transfectedDAMI cells taken at different times after mixing were treated withanti-c-src antibodies, followed by protein-G sepharose beads. To thebeads was then added γ³²PATP; subsequently the proteins were solubilizedfrom the beads, and subjected to SDS17 PAGE and autoradiography. Theresults in FIG. 9b demonstrate that several transient bands appear thatare maximally phosphorylated at 8-10 min after cell mixing, a timecourse corresponding to the appearance of PTyr in the analogous extracts(FIG. 9a , Panel 1). Prominent among these bands is one doubletcorresponding to c-Src, confirming that c-Src is activated transientlyin the β-APP:PS-1 intercellular interaction.

The identities of the other phosphorylated bands in FIG. 9b are notknown. Not all of them are necessarily due to tyrosine phosphorylation;some serine or threonine kinases might have been bound to the c-Src thatwas immuno-reacted with specific anti-pc-Src. Involvement of Lyn but notFyn downstream of β-APP:PS-2 intercellular signaling. When β-APP:PS-2intercellular interactions were carried out with mixtures ofappropriately transfected DAMI cells, an entirely different set ofproteins was PTyr modified than for the β-APP:PS-1 system. Althoughbands were detected by the PTyr antibody that were present at 50-66 kDa,these did not correspond to c-Src on Western blots (FIG. 11a , Panel 1).Furthermore, when extracts of β-APP:PS-2 mixed cells were firstimmunoprecipitated with c-Src antibodies and the immunoprecipitates werethen autophosphorylated in vitro, no significant increases inphosphorylation at the earlier time points (β-10 mins after mixing) wereseen (FIG. 10b ).

At later time points however, c-Src could apparently be phosphorylatedin these samples indicating that it contributes to increases identifiedin the second later peak of β-APP:PS-2 signaling (FIG. 10b ). Thepossible involvement of other members of the Src kinase family wasinvestigated with molecular weights in the 53-59 kDa range other thanc-Src. Lyn (Mwt 53/56 kDa) and Fyn (Mwt 59 kDa) were two candidate Srckinases that were examined.

Results of Western blot hybridization with anti-Lyn antibodies in FIG.11a show that Lyn protein concentrations do not change when β-APP:PS-2intercellular interactions are carried out, but afterimmunoprecipitation of the extracts with anti-Lyn antibodies and invitro autophosphorylation of the precipitates, a transientphosphorylation of Lyn with peaks of activity at 8-9 and 17-18 min isobserved, along with other phosphorylated bands (FIG. 11c ). Lynundergoes transient phosphorylation in a pattern that is similar to thePTyr increases seen on Western blots and ELISAs for β-APP:PS-2interaction (FIG. 11c ). Fyn, on the other hand, shows noautophosphorylation in-vitro in the same extracts afterimmunoprecipitation with anti-Fyn antibodies (FIG. 11d ), nor any changein its concentration with time (FIG. 11b ).

Example 3

The following data demonstrates G-protein binding to endogenous PS-1 andPS-2 in extracts of mouse frontal cortex. A 20% homogenate of WT mousefrontal was made in GTPγS solublization/extraction buffer [50 mMHEPES/NaOH pH 7.4, 1 mM EDTA, 1 mM DTT, 1% Triton-X100, 60 mMoctylglycoside, 1× Protease inhibitor mix (1 uMphenylmethylsulfonylflouride, 1 ug/mL antipain, 0.1 ug/mL pepstatin A,0.1 ug/mL leupeptin)]. Measurements of [³⁵S]GTPγS binding were performedon Untreated, PTX treated; and PS-1 and PS-2 immuno-depleted extracts.

For untreated samples, 100 μg of extract was brought up to 100 uL inGTPγS solublization/extraction buffer and mixed with an equal volume ofGTPγS Buffer B (50 mM HEPES/NaOH pH 7.4, 40 μM GDP, 50 mM MgCl₂, 100 mMNaCl) for a total volume of 200 μL. The reaction was started with 50 nM[³⁵S] GTPγS (1250 Ci/mMol; Perkin Elmer) and incubated at RT for 60 min.The reaction was stopped by addition of 20 uL 10× Stopping buffer (100mM Tris-HCl, pH 8.0, 25 mM MgCl₂, 100 mM NaCl, 20 mM GTP).

For PTX treated samples, 100 μg of extract was brought up to 100 μL inGTPγS solublization/extraction buffer and treated with 500 ng/mLactivated PTX in the presence of PTX Buffer (20 mM HEPES pH 8.0, 1 mMEDTA, 2 mM MgCl₂, 1 mM NAD). The sample was incubated for 12 hrs at 30°C. The PTX treated sample was then mixed with an equal volume of GTPγSBuffer B and taken through [³⁵S]GTPγS assay as described above.

Extracts of mouse frontal cortex were immunoprecipitated with a mixtureof polyclonal antibodies to PS-1 and PS-2 (10 uL each) at 4° C.overnight to deplete the samples of PS-1 and PS-2. Protein A agarose (20uL slurry/100 μg protein) was added and samples were and shakenend-over-end at 4° C. for 2 h. The PS-antibody-protein A complex wascentrifuged at high speed for 5 min. Supernatant was recovered in and100 μg aliquots were taken through the [³⁵S] GTPγS assay as described.

Following the GTPγS reaction, 5 uL of either anti-PS-1 or anti-PS-2monoclonal antibodies were added and samples were placed at 4° C.overnight. The antibody-protein complex was bound to 20 μL Protein A/Gagarose (Pharmacia) and samples were placed at 4° C. and shakenend-over-end for 2 hrs. The agarose beads were washed three times withWash Buffer 1 (50 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 1% Triton-X100,1× protease inhibitor mix) and once with each Wash Buffer 2 (50 mMHEPES, pH 7.4, 1 mM EDTA, pH 8.0, 0.5% Triton-X100, 1× proteaseinhibitor mix) and 3 (50 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 1×protease inhibitor mix). The washed agarose beads were then suspended in5 mLs scintillation fluid (CytoScint, ICN) and counted on a BeckmanCoulter LS 6000 SC scintillation counter for 3 min.

FIG. 12 shows the ³⁵S-GTPγS incorporation in extracts of mouse brainthat could be immunoprecipitated with monoclonal antibodies to PS-1,suggesting the co-precipitation of ³⁵S-GTPγS-bound G-protein with theendogenous PS-1. This incorporation was greater than 80% of that foundfor extracts which had been prior depleted of PS-1 and PS-2 by treatmentwith polyclonal antibodies to the two PS proteins, showing specificityof the G-protein:PS-1 binding. Treatment with PTx inhibited the³⁵S-GTPγS incorporation by 60%.

FIG. 13 shows the ³⁵S-GTPγS incorporation in extracts of mouse brainthat could be immunoprecipitated with monoclonal antibodies to PS-2,suggesting the co-precipitation of ³⁵S-GTPγS-bound G-protein with theendogenous PS-2. This incorporation was greater than 85% of that foundfor extracts which had been prior depleted of PS-1 and PS-2 by treatmentwith polyclonal antibodies to the two PS proteins, showing specificityof the G-protein:PS-2 binding. Treatment with PTx inhibited the³⁵S-GTPγS incorporation by 55%. These results demonstrate a specificPTx-sensitive G-protein coupling to endogenous mouse brain PS-1 andPS-2.

Sequences corresponding to the first 16 amino acids of intracellularloop 1 [ic1(1-16)], the remaining 16 amino acids of intracellular loop 1[ic1(17-32)], the entire intracellular loop 2 (ic2), the entireintra-cellular loop 3 (ic3), the first 20 amino acids of the cytoplasmicC-terminal tail (C1-20) and the remaining 19 amino acids of thecytoplasmic C-terminal tail (C21-39) for both PS-1 and PS-2 will besynthesized and HPLC purified to >90% purity. FIG. 14 illustratesintracellular domains of PS. Table 1 shows the sequences that can besynthesized from these domains. In addition, a 20 amino acid controlpeptide synthesized in which the sequences of peptide C1-20 can bescrambled. This peptide is part of the 39 amino-acid sequence identifiedas the binding domain on PS-1 for G_(o).

TABLE 1 PS-1 Cytoplasmic PS-2 Cytoplasmic peptides peptides ic1(1-16)KSVSFYTRKDGQLIYT KSVRFYTEKNGQLIYT (SEQ 2 aa 101-115) (SEQ 4 aa 107-122)ic1  PFTEDTETVGQRALHS TFTEDTPSVGQRLLNS (17-32) (SEQ 2 aa 117-132)(SEQ 4 aa 124-138) ic2 VFKTYNVAVD EVLKTYNVAMD (SEQ 2 aa 185-194)(SEQ 4 aa 190-200) ic3 KYLPE (identical to (SEQ 2 aa 239-243) ic3 of PS-1) C(1-20) KKALPALPISITFGLVFYFA KKALPALPISITFGLIFYFS(SEQ 2 aa 429-448) (SEQ 4 aa 410-429) C(21-39) TDYLVQPFMDQLAFHQFYITDNLVRPFMDTLASHQLYI (SEQ 2 aa 449-467) (SEQ 4 aa 430-448)

Example 4

The present studies demonstrate that the GPCR function of PS-1 modulatesthe production of Aβ. A major question in the study of PS-GPCR functionis to determine a specific ligand for PS that can elicit G-proteinactivities from the PS, to which the ligand binds intercellularly. Thepresent studies investigated whether the three-partligand-receptor-G-protein system initiates the production of Aβ. In sucha system activation of PS by ligand (β-APP) binding would lead toG-protein binding to PS in the cytoplasmic domain.

In order to investigate whether G-protein binding to PS-1 or PS-2affects Aβ production from β-APP, cell:cell interaction of β-APP andPS-1 in the presence and absence of Pertussis toxin (PTx) experimentswere performed. PTx is a specific inhibitor of G-protein G_(o)activation. If the GPCR function of PS is involved in the production ofAβ from β-APP:PS intercellular binding, then in its presence, Aβproduction should be inhibited.

β-APP:PS-1 mediated cell-cell interactions were carried out usingmethods described above, with PS-1 transfected primary β-APP−/−fibroblasts (cells express PS-1 and do not produce β-APP) interactedwith β-APP-transfected ES (PS−/−) cells (cells produce β-APP but do notexpress PS) in the presence of ³⁵S-methionine. 24 h after co-culture ofthe transfected cells, the samples were harvested in the presence ofprotease inhibitors. Cells were sonicated and 100 μg of whole cellextracts were immunoprecipitated with antibodies to Aβ (6E10) andimmunoprecipitated samples were run on Bicene-Tris gels. Aβ bands werevisualized by autoradiography of dried gels. The same experiment wascarried out in the presence of 500 ng/ml of PTx. Treatment of culturedcells was carried out for 12 h as described below. As a control for PTxtreatment, the cultured cells were incubated with PTx buffer onlycontaining ATP and NAD. Under these conditions activation of Go andlevels of Aβ should be unaffected.

FIG. 15 shows the results of these studies. Lane 1 shows the results ofβ-APP-expressing ES (PS−/−) cells co-cultured with PS-1-expressingFibroblasts (β-APP−/−). Lane 2 shows the results of the components usedin lane 1 in the presence of PTx and PTX buffer (NAD+ATP). Lane 3 showsthe results of the components used in lane 1 in the presence of PTxbuffer only (NAD+ATP), and no PTx. Lane 4 shows the results of tail-lessβ-APP-expressing ES (PS−/−) cells co-cultured with +Tail-lessPS-1-expressing Fibroblasts (β-APP−/−). Lane 5 shows the results of thecomponents used in lane 4 in the presence of PTx. Lane 6 shows theresults of wild type β-APP-expressing ES (PS−/−) cells co-cultured withTail-less PS-1-expressing fibroblasts (β-APP−/−).

The results indicate that PTx toxin inhibits the production of Aβ fromthe intercellular interaction of β-APP and PS-1 (lanes 1 and 2 above).Lane 3 shows that in the presence of PTx buffer only, but in the absenceof PTx, Aβ production is not inhibited. Lanes 4 and 6 show that thecytoplasmic carboxyl terminal domain of PS-1, earlier shown to be thebinding domain of PS-1 for Go, when absent, eliminates Aβ production.

The data provided herein indicate that β-APP is a ligand for PS-1 whichupon binding activates its GPCR activity. The data also indicates thatthe GPCR function of PS-1 is involved in the production of Aβ from β-APPafter its intercellular interaction with PS-1. These results furtherindicate that modulating GPCR activity of PS-1 also modulates theproduction of Aβ. Accordingly, agents that modulate GPCR activity ofPS-1 will modulate the production of A13.

For co-culture experiments ES (PS^(−/−)) and β-APP(^(−/−)) cells wereplated at 1×10⁷ cells per 25 cm² flask and transfected with theappropriate cDNAs. 5 hours after transfection, ES(PS-1^(−/−)/PS-2^(−/−)) cells transfected with β-APP were detached bymild trypsinization, washed 2× with met-free culture medium containingheat-inactivated, dialysed FCS (10% v/v) and resuspended in this mediumat 0.33×10⁷ cells/ml. Similarly, primary fibroblasts from β-APP knockoutmice were co-transfected with PS-1 or PS-2 and plated at 1×10⁷ cells.Transfected cells were washed 2× with met free medium and left in 3 mlmet-free medium.

β-APP transfected ES (PS-1^(−/−)/PS-2^(−/−)) cells (1×10⁷ cells/3 mlmet-free medium) were added to the PS-1-transfected β-APP knockoutcells. The cell densities ensured that essentially all the cells were incontact with another. ³⁵S-met (66 μCi/ml; 1175 Ci/mmol, NEN) was addedand the cultures incubated for 24 h. In experiments with PTx treatment,500 ng/ml PTx was added to the cultures under the appropriate reactionconditions at this stage and incubated for 24 h. The medium was thenremoved and cells harvested by scraping. A protease inhibitor mix wasadded to the medium before freezing on dry ice. 100 μl extraction buffer(50 mM Tris, pH 8.0/150 mM NaCl/0.5% Nonidet-P40) containing proteaseinhibitors (1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride(AEBSF)/1 μg/ml antipain/0.1 μg/ml pepstatin A/0.1 μg/ml leupeptin) wasadded to the cell pellet and the samples quick-frozen on dry ice.

The PTx protomer (Biomol Research Laboratories) was incubated with 10 mMDTT at 37° C. for 10 min to convert it to its enzymatically active form.5 h after transfecting ES cells with PS-1 or PS-2 and the G-proteincDNAs, 500 ng/ml of activated PTx was added to the cells in culturemedium in the presence of 1 mM NAD, 1 mM ATP, 2 mM MgCl₂ and 1 mM EDTA.The cells were incubated at 37° C. in the presence of 5% CO₂ for 18 h.

Whole cell extracts were prepared using cell-pellets sonicated with 3bursts of 20 seconds each on ice. Protein concentration was determinedaccording to the method of Lowry.

Immunoprecipitations were carried out using 100 μg of cell extractsubjected to immunoprecipitation in an end-over-end rotator at 4° C.overnight with 2 μg Aβ specific monoclonal antibodies 6E10 (Senetek),which was raised to residues 1-17 of Aβ (Senetek). 40 μl slurry ofProtein G sepharose (Pharmacia) was then added and allowed to mixend-over-end for 1 h at room temperature. The antigen-antibody-Protein Gsepharose complex was washed once with each of the following: buffer 1(10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 0.65M NaCL, 1% NP-40),buffer 2 (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 0.75% NP-40) andbuffer 3 (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 0.1% NP-40). Thewashed complex was boiled for 10 min in bicene-tris sample buffer andsubjected to SDS PAGE on bicene-tris gels.

Bicene-tris gels (15% T/5% C) with 8M urea was cast and run. The gelswere then fixed for 30 min with 5% glutaraldehyde in 0.4M sodiumborate/phosphate buffer and stained for 1 h with 0.1% Coomassie BlueG250 in methanol-acetic acid. After destaining the gels were preparedfor autoradiography.

The destained gels were treated with ethanol (30%) and glycerol (5%) for30 min and impregnated with Amplify (Amersham) for 30 min, dried undervacuum at 80° C. and exposed to X-Omat film at −70° C. for 4-5 days.

The specific β-APP:PS-mediated cell-cell interaction that is requiredfor the eventual production of Aβ also activates G-protein. Cell:cellinteraction, mediated by the specific binding of β-APP on one cellsurface with PS on the other cell surface, is a required initial step inthe production of Aβ from β-APP. To determine whether a cell-cellinteraction between human β-APP-only- and PS-only-expressing cells,along with their associated endogenous proteins, resulted in endogenousmouse G-protein activation, binding of ³⁵S-GTPγS with Gα, a standardmethod to determine G-protein activation, was assayed. In all of thesestudies, activation of endogenous mouse Gα_(o) was examined.

Two cell types were generated: ES-derived mouse cells (PS-1^(−/−);PS-2^(−/−)) that expressed only small amounts of endogenous β-APP but noPS, were transiently transfected with cDNA for human β-APP to producecells expressing excess human β-APP over mouse β-APP, and no PS-1 orPS-2 (β-APP-only cells). Embryonic (E18) mouse primary fibroblastsderived from β-APP-null mice, expressing only small amounts ofendogenous PS-1 and PS-2, were transfected with cDNAs for human PS-1 toproduce cells expressing excess human over mouse PS-1 but no β-APP(PS-1-only cells). The β-APP-only and PS-1-only expressing cells wereco-cultured for 24 h, either at high densities which allowed theirintercellular interaction, or at lower densities which permitted onlylesser degrees of cell:cell interaction (see the light micrographs inFIG. 16C, lane 2 compared to FIG. 16C, lane 4).

Detergent-buffer extracts of the high and low-density co-cultures in thepresence and absence of Pertussis toxin (PTx) (the activation of G_(o)is inhibited in the presence of PTx) were prepared and aliquots eachcontaining 100 μg total protein were reacted with ³⁵S-GTPγS.Immunoprecipitation of each ³⁵S-GTPγS-treated extract was carried outwith polyclonal antibodies K-20 directed to Gα_(o), whichimmunoprecipitated the activated ³⁵S-GTPγS-labeled mouse Gα_(o).

Where β-APP-only cells were co-cultured with PS-1-only cells at highdensity (FIG. 16C, lane 1), there was a greater than 200% increase in³⁵S-GTPγS incorporation (FIG. 16A, lane 1) over basal values obtainedfrom control extract (prepared by mixing equal parts of untransfected ES(PS^(−/−)) and fibroblast (β-APP^(−/−)) cell extracts), compared to a33% increase (FIG. 16A, lane 2) for the low density samples (FIG. 16C,lane 2). In the presence of PTx, the high density cultures (FIG. 16C,lane 4) showed an inhibition in ³⁵SGTPγS incorporation, 38% lower thanbasal values (FIG. 16A, lane 3). These results indicate that a specificcell:cell interaction between the β-APP-only and PS-only cells alsoactivates PTx-sensitive G-proteins either directly via PS-1 cytoplasmicdomains, or indirectly via PS-associated proteins.

β-APP-only and PS-only cells were co-cultured at high and low densitiesand metabolically labeled in the presence of ³⁵S-met, either with orwithout PTx. After 24 h co-culture, the cells were harvested anddetergent extracts prepared. Samples, each containing 100 μg protein,were immunoprecipitated with mouse MAb (6E10) to human Aβ, followingwhich the solubilized immunoprecipitates were electrophoresed onBicene-Tris gels. The Aβ bands were then visualized by³⁵S-autoradiography. FIG. 16 demonstrates: 1) that Aβ levels present inequal amounts of total protein decreased steadily (FIG. 16B, lanes 1-3)with decreasing cell density (FIG. 16C, lanes 1-3), consistent with arequirement for a β-APP:PS-1-mediated intercellular binding in order toproduce Aβ from β-APP; 2) a requirement for the same intercellularinteraction for PTx-sensitive G-protein activation, as shown in FIG.16A; and 3) that the production of Aβ in the high density culture (FIGS.16B, lane 1 and 16C, lane 1) was completely inhibited in the presence ofPTx (FIGS. 16B lane 4 and 16C lane 4). The demonstration of aninhibition of both G-protein activation and Aβ production by PTxstrongly suggests that the activation of the G-protein occurs prior toAβ formation, consistent with the concept that such activation is on thepathway to Aβ production from the β-APP:PS-1 intercellular binding.

A fusion construct of the water-soluble entire 80 amino acidNH₂-terminal domain of PS-1 (Peptide 1-80) added to the co-culturemedium functioned as a specific competitive inhibitor of the β-APP:PS-1mediated cell-cell interaction and inhibited Aβ production. As a furtherevidence of a requirement for a specific cell-cell interaction betweenβ-APP and PS for both G-protein activation and Aβ production,experiments were performed to determine whether the activation of Gα_(o)that resulted from this cell-cell interaction could be inhibited by theaddition to the co-cultures of Peptide 1-80.

Co-cultures of β-APP-only and PS-1-only cells were carried out in thepresence and absence of Peptide 1-80 (0-3 μM). ³⁵S-GTPγS assays werecarried out on extracts as a measure of G-protein activation. The Aβproduced in these co-culture extracts was determined by ELISA. FIG. 17shows that both, the production of Aβ (FIG. 17A) and the activation ofGα_(o) (FIG. 17B), could be inhibited by Peptide 1-80 in adose-dependent manner, consistent with the view that the G-proteinactivation results from the same β-APP:PS-1 mediated cell-cellinteraction that initiates the eventual production of A8.

In addition, water-soluble β-APP ectodomain itself (β-APPs), when addedto cultures of PS-1-only cells, could also activate G-proteins in adose-dependent manner. β-APPs was partially purified from conditionedmedia of baculovirus cultures over-expressing human β-APP. The finalproduct (FIG. 18b , lane 1) had, in addition to β-APP, three majorcontaminating bands at 81 kDa, 55 kDa and 31 kDa that co-purified withthe β-APP ectodomain. This partially purified β-APPs preparation wasadded in increasing amounts to PS-1-only APP^(−/−) fibroblasts. After 15mins the cells from each well were harvested in extraction buffer, anddetergent extracts (each containing 100 μg protein) were treated with³⁵S-GTPγS. Activation of Gα_(o) was assayed after immunoprecipitation ofthe activated ³⁵S-GTPγS-Gα_(o) with the Gα_(o)-specific antibodies.Increase in incorporation of ³⁵S-GTPγS (FIG. 3A, curve 1) was observedin the PS-1-only cells with increasing concentration of the partiallypurified β-APPs, with a maximum increase of over 500% with the additionof 120 pM β-APPs. Cultures of untransfected APP^(−/−) fibroblasts gavemodest G-protein activation (FIG. 18A, curve 3), presumably due to thepresence of endogenous PS-1 in these cells. Furthermore, when partiallypurified β-APPs was added to untransfected ES (PS^(−/−)) cells, noincrease in G-protein activation was observed by ³⁵S-GTPγS assays (FIG.18A, curve 5), further implicating PS as a receptor for the β-APP ligandin the Gα_(o) activation.

To confirm that it was indeed the β-APPs in the partially purifiedβ-APPs preparation that was the ligand responsible for G-proteinactivation, and not one of the contaminants, β-APPs was removed from thepreparation by treatment with MAb 348 to β-APP (see FIG. 18C, lane 2).This β-APPs-depleted solution at the same concentrations used for thenon-depleted preparation, was added to the PS-1-only cells, which werethen treated exactly as described above. FIG. 18A (curve 4) shows thatthe antibody removal of β-APPs resulted in a nearly total loss of the³⁵S-GTPγS incorporation that was observed with the β-APPs preparation.In contrast, the β-APPs preparation similarly treated with an irrelevantIgG when added to PS-1-only cells, gave results that resembled thoseobtained with the untreated samples (FIG. 18A, curve 2). This result isconsistent with the proposal that the loss of G-protein activation (FIG.18A, curve 4) was not due to non-specific protein loss during theimmunodepletion procedure, but was due to the specific immuno-removalfrom the sample of the β-APPs. These results provide evidence thatmembrane-detached water-soluble β-APPs itself, in addition to intactmembrane-bound β-APP, can induce G-protein activation in the PS-1expressing cells, but not in the absence of PS.

Previous reports have shown that G-protein G_(o) also binds to β-APP. Inthe experiments just described with β-APPs there were no β-APPcytoplasmic or intra-membrane domains present; the experiments utilizedβ-APP^(−/−) cells transfected with PS, to which only the solubleectodomain of β-APP was added. Thus the G_(o) activation observed in allthe work described herein is due specifically to G_(o) activation onlyat the PS cytoplasmic domain, or that of a PS-associated protein.

Because the results so far described were obtained using only culturedcells transfected with human PS, binding of the endogenous rat PSproteins with rat Gα_(o) present in rat hippocampal membranes wasexamined. These membranes were solubilized and ³⁵S-GTPγS assays werecarried out on the solubilized membranes, both before and after theaddition of increasing concentrations (0-120 pM) of the partiallypurified β-APPs. FIG. 19A shows that at the lowest concentration ofβ-APPs (80 pM), the incorporation of ³⁵S-GTPγS was 100% greater thanwith untreated samples, and at 400 pM β-APPs, G-protein G_(o) activationincreased by greater than 600%. These increases (FIG. 19B) did not occurif the solubilized rat membranes were first treated with a mixture ofpolyclonal Ab directed to both PS-1 and PS-2 to deplete the samples ofthese two mouse proteins, consistent with the participation of PS-1, orits associated proteins, in the G-protein activation by β-APPs.

Experiments were performed to determine whether the human G-proteinG_(o) binds to intact human PS-1 inside ES-derived (PS^(−/−)) mousecells that had been variously transfected with cDNA for human PS-1together with the cDNA for either human Gα_(oA) or Gα_(oB) proteins.Detergent extracts were prepared from these cells and examined. Extractseach containing 100 μg of protein from ES cells that had been variouslytransfected, were first immunoprecipitated with MAb specific for thelarge hydrophilic loop of PS-1 (which protrudes from the exterior of theplasma membrane in the 7-TM model of PS). The immunoprecipitates werethen solubilized and subjected to SDS-PAGE, followed by Western blothybridization with Ab K-20 to Gα, that recognizes both isoforms, Gα_(oA)and Gα_(oB). Only the PS-1/Gα_(oA) co-transfected cells gave a robustsignal for Gα_(o) at ˜40 kDa (FIG. 20A, lane 3 compared to lanes 2 and4), suggesting that Gα_(oA), but not Gα_(oB), binds specifically to PS-1(the binding being retained in the detergent solution used). The resultsreflecting the in vitro selective binding of human PS-1 and Gα_(o) arein agreement with the published studies of the in silico binding ofhuman Gα_(o) to human PS-1, but they further distinguish between the twoGα_(o) isoforms, a result not previously shown.

To provide evidence that the G_(oA) binding observed in FIG. 5A wasdirectly to PS-1, and not to a co-immunoprecipitated, PS-associatedprotein the autonomous activation of G_(o) by synthetic peptidefragments corresponding to cytoplasmic loops 1, 2, 3 and the carboxyltail of PS-1 (in the 7-TM PS model) were investigated (see FIGS. 20B andC and Table 1). The peptides were individually tested for their abilityto stimulate ³⁵S-GTPγS binding to G_(o) in rat hippocampal membranepreparations.

In FIG. 20C evidence is presented that cytoplasmic loop 1 and 2 peptides(FIG. 20B) induced only background stimulation of ³⁵S-GTPγS binding.Cytoplasmic Loop 3 peptide however, produced a robust stimulation of³⁵S-GTPγS binding to G_(o), as did a peptide corresponding to the first20 amino acids of the COOH-tail. A second COOH-terminal peptidecorresponding to residues 21-39, however, failed to activate G_(o). Theresult with the peptide corresponding to residues 1-20 of the COOH-tailare in keeping with G-protein activation by the COOH— terminal domain ofPS-1. That study, however, missed G_(o) activation by cytoplasmic loop 3of 7-TM PS-1, a region known to be important for G-protein binding formost GPCRs. The results confirm that the G_(oA) binds directly to PS-1,and not to PS-associated proteins.

As to the G-protein activation following β-APP:PS cell-cell interaction,it may be involved in Aβ production in any of a number of ways. It maysignal the phosphorylation or internalization of the β-APP:PS complexfrom the plasma membrane, or it may activate other downstream eventssuch as Ca²⁺ release, that might be directly involved in the pathway tothe production of Aβ.

The data demonstrate that the G-protein activation resulting from thespecific cell:cell interaction of β-APP with PS is on the pathway of thesubsequent production of Aβ from β-APP.

In order to determine whether any other G-proteins besides Gα_(o) cancouple the Presenilin (PS)-1 cytoplasmic domains, Gα COOH Minigenevectors were used. The carboxyl terminal domains from various G-proteinGα subunits are important sites for receptor binding, and peptidescorresponding to the COOH-termini can be used as competitive inhibitorsof receptor-G-protein interactions. Minigene vectors that encode 11-14C-terminal amino acids of Gα have the ability to inhibit/block receptormediated activation of signaling pathways. Minigenes cloned in pcDNA3were obtained from Caden Biosciences. Table 2 shows the specificsequences for each minigene used.

TABLE 2 Peptide Sequences encoded by  Gα Minigene vectors Gα InsertC-Terminal Peptide Sequence Gα_(i1/2) IKNNLKDCGLF (SEQ ID NO: 18)Gα_(i3) IKNNLKECGLY (SEQ ID NO: 19) Gα_(oA) IANNLRGCGLY (SEQ ID NO: 20)Gα_(oB) IAKNLRGCGLY (SEQ ID NO: 21) Gα_(z) IQNNLKYIGLC (SEQ ID NO: 22)Gα_(s) QRMHLRQYELL (SEQ ID NO: 23) Gα_(olf) QRMHLKQYELL (SEQ ID NO: 24)Gα_(q) LQLNLKEYNAV (SEQ ID NO: 25)

ES-derived mouse cells (PS-1^(−/−); PS-2^(−/−)) that expressed onlysmall amounts of endogenous β-APP but no PS, were transientlytransfected with cDNA for human β-APP to produce cells expressing excesshuman β-APP over mouse β-APP, and no PS-1 or PS-2 (β-APP-only cells).Embryonic (E18) mouse primary fibroblasts derived from β-APP-null mice,expressing only small amounts of endogenous PS-1 and PS-2, weretransfected with cDNAs for human PS-1 to produce cells expressing excesshuman over mouse PS-1 but no β-APP (PS-1-only cells). The minigenes wereco-transfected with PS-1 in the PS-only APP−/− fibroblasts to inhibitthe endogenous mouse Gα under investigation in those cells. PS−/−EScells were separately transfected with human β-APP cDNA (β-APP onlycells) and the two types of cells were co-cultured for 24 hours. Cellswere harvested and detergent-buffer extracts of the co-cultures wereprepared. Determination of [³⁵S]GTPγS incorporation in these cultureswas carried out in the presence and absence of each minigene: Aliquotseach containing 100 μg total protein were reacted with ³⁵S-GTPγS andimmunoprecipitation of each ³⁵S-GTPγS-treated extract was carried outwith antibodies directed to the particular Gα under investigation.Specificity of binding of a particular Gα to PS-1 was established byblocking of GTPγS incorporation in the presence of the specific minigeneinhibitor.

FIG. 23 shows the presence or absence of G-protein activation, followingβ-APP:PS-1 cell-cell interaction, of Gα_(oA), Gα_(B), Gα_(q), Gα,Gα_(i1/2) and Gα_(z) in the presence and absence of their specificminigene inhibitors. β-APP:PS-1 cell-cell interaction specificallyactivated Gα_(oA), Gα_(q) and Gα_(s), and that activation was inhibitedby the specific minigene inhibitor of each G-protein. The presence ofthe Gα_(oB) minigene did not inhibit the activated Gα_(o) proteinimmunoprecipitated by the Gα_(o) antibody (this antibody cross-reactswith both the G_(oA) and G_(oB) isoforms) suggesting that the activatedspecies is the G_(oA) protein in this sample, which is not inhibited bythe G_(oB) minigene. No G-protein activation was seen for Gi1/2 or Gz asa result of the β-APP:PS-1 cell-cell interaction, suggesting that theseG-proteins do not couple PS-1.

These data demonstrate that cell-cell interaction between β-APP and PS-1also activates G-proteins G_(q) and G_(s). Aβ produced in the co-cultureextracts described above were determined by ELISA. The results in FIG.24 show that for the PS-1:βAPP intercellular interaction, the activationof Gα_(oA) and Gα_(q) coincides with the production of Aβ, whereas theactivation of Gα_(s) does not. When the G-protein activation of theseproteins is inhibited by the presence of the specific minigene inhibitorfor each G-protein, the Aβ production is also inhibited in the case ofGα_(oA) and Gα_(q), but not in the case of Gα_(s). These resultsdemonstrate that the production of Aβ from β-APP involves the downstreamsignaling pathways of the G-proteins G_(o) and G_(q), both of whichsignal via PLC (different mechanisms).

The autonomous activation of G-proteins Gα_(o), Gα_(q) and Gα_(s) byoligopeptides corresponding to cytoplasmic loops 1, 2, 3 and the COOHtail of human PS-1 in the 7-TM PS model were examined. The peptides wereindividually tested for their ability to stimulate ³⁵S-GTPγS binding toeach G-protein in rat hippocampal membrane preparations.

FIG. 25A shows the autonomous activation of Gα_(o) by cytoplasmic looppeptides of PS-1. Evidence is presented that cytoplasmic loop 1 and 2peptides induced only background stimulation of ³⁵S-GTPγS binding.Cytoplasmic Loop 3 peptide however, produced a robust stimulation of³⁵S-GTPγS binding to G_(o), as did a peptide corresponding to the first20 amino acids of the COOH-tail. A second COOH-terminal peptidecorresponding to residues 21-39, however, had no detectable G_(o)activation.

FIG. 25B shows the autonomous activation of Gα_(q). Cytoplasmic loop 1and 2 peptides induced only background stimulation of ³⁵S-GTPγS binding.Cytoplasmic Loop 3 peptide, as for Gα_(o), produced a robust stimulationof ³⁵S-GTPγS binding to G_(q), as did a peptide corresponding to thelast 19 amino acids of the COOH-tail. A COOH-terminal peptidecorresponding to residues 1-20, previously shown to activate G_(o), didnot provide detectable activation of G_(q).

FIG. 25C shows the autonomous activation of Gα_(s). Cytoplasmic loop 1peptides comprising residues 17-32 (but not the first 16 amino acids ofloop 1) as well as a peptide corresponding to the last 19 amino acids ofthe COOH-tail induced stimulation of ³⁵S-GTPγS binding. CytoplasmicLoops 2 and 3 peptides, as well as the COOH-terminal peptide 1-20 forGα_(o) induced only background stimulation of ³⁵S-GTPγS binding.

These results indicate that the different G-proteins bind to differentcytoplasmic domains or combination of domains on the PS-1.

Primary Antibodies: Polyclonal Ab to Gα_(o), K-20, sc-387, Gαq, Gαs,Gαi1/2, and Gαz were purchased from Santa Cruz Biotechnology. Gα_(o) Abrecognizes both, G_(oA) and G_(oB). MAb 6E10 (Senetek), to residues 1-17of Aβ recognizes both, full-length β-APP, as well as Aβ.

Cell culture: Mouse ES (PS-1^(−/−)/PS-2^(−/−)) were cultured aspreviously described.

Preparation of Whole Cell Extracts: Extracts were prepared by sonicationin solublilization buffer 1 (50 mM HEPES/NaOH pH 7.4, 1 mM EDTA, 1 mMDTT, 1% Triton-X100, 60 mM octylglycoside, 1× Protease inhibitor mix)and proteins using a Lowry assay.

³⁵S-GTPγS Assays: Extract (100 μg of total protein) was mixed with anequal volume of GTPγS Buffer B (50 mM HEPES/NaOH pH 7.4, 40 μM GDP, 50mM MgCl₂, 100 mM NaCl) and reacted with 50 nM ³⁵S-GTPγS (1250 Ci/mmol,Perkin Elmer, Waltham, Mass.) for 60 min at RT. The reaction was stoppedwith 10× Stopping buffer (100 mM Tris-HCl, pH 8, 25 mM MgCl₂, 100 mMNaCl, 20 mM GTP) followed by immunoprecipitation of the ³⁵S-GTPγS-Gα_(o)complex with Ab against a particular Gα. The Ab-protein complex wasabsorbed to Protein A/G agarose for 90 min at RT and washed. The agarosebeads were suspended in scintillation fluid (CytoScint, ICN) (5 ml) andcounted in a Beckman Coulter LS 6000 SC scintillation counter for 3 min.

Co-culture of β-APP-only with PS-1-only cells was carried out asdescribed above.

ELISA for the production of A131-40 was carried out using sandwich ELISAkits (Biosource).

Rat hippocampal membranes (Applied Cell Science, Rockville, Md.) weresolubilized in CHAPs buffer.

Peptides (200 μM) were incubated without pre-treatment with solubilizedrat hippocampal membranes (50 μg) in the ³⁵S reaction mixture.Accumulation of γ-S GTP the ³⁵S-GTPγS was determined afterimmunoprecipitation with the specific anti-G Ab.

TABLE 3 Binding of different G-Proteins to Human PS-1 G-Pro-PS-1 Cytoplasmic PS-1 tein Binding Sequence Residues Gα_(o) KYLPE239-243* KKALPALPISITFGLVFYFA 429-448* Gα_(q) KYLPE 239-243*TDYLVQPFMDQLAFHQFYI 449-467* Gα_(s) PFTEDTETVGQRALHS 117-132*TDYLVQPFMDQLAFHQFYI 449-467* *Residue numbers are with respect to SEQ IDNO: 2

Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the teachings of the disclosure or the scope of thedisclosure as defined by the appended claims. The appendices attachedhereto are provided to further illustrate but not limit the invention.

What is claimed is:
 1. A pharmaceutical composition comprising an isolated polypeptide in a pharmaceutically acceptable carrier, wherein the polypeptide consists essentially of an amino acid sequence from the N-terminal fragment of PS-1 (SEQ ID NO:2), wherein the fragment is selected from the group consisting of: (i) DEEEDEEL (SEQ ID NO:5), (ii) SEQ ID NO:5 having 1-10 additional amino acids at either the N- and/or C-terminal end(s), (iii) RRSLGHPEPLSNGRPQGNSRQWEQDEEEDEELTLKYGAK (SEQ ID NO:7), (iv) a polypeptide that is at least 85% identical to SEQ ID NO:7 having 1-5 conservative amino acid substitutions, (v) a sequence of (iii) or (iv) having 1-10 additional amino acids at the N- and/or C-terminal end(s), (vi) DEEEDELTLKYGAK (SEQ ID NO:17), (vii) SEQ ID NO:17 having 1-2 conservative amino acid substitutions, (viii) a sequence of (vi) or (vii) having 1-10 additional amino acids at the N- or C-terminus, (ix) any of the foregoing polypeptides comprising an unnatural amino acid or D-amino acid, and (x) combinations of any of the foregoing, wherein the isolated polypeptide inhibits production of Aβ in a cell.
 2. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence DEEEDEEL (SEQ ID NO:5).
 3. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence RRSLGHPEPLSNGRP (SEQ ID NO:6).
 4. The pharmaceutical composition of claim 1, wherein the polypeptide comprises an unnatural amino acid or D-amino acid.
 5. The pharmaceutical composition of claim 1, wherein the composition includes a sterile aqueous solution.
 6. The pharmaceutical composition of claim 1, wherein the composition is in a solvent-free form.
 7. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence of DEEEDEEL (SEQ ID NO:5) having 1-10 additional amino acids at either the N- and/or C-terminal end(s).
 8. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence RRSLGHPEPLSNGRPQGNSRQWEQDEEEDEELTLKYGAK (SEQ ID NO:7).
 9. The pharmaceutical composition of claim 8, wherein the polypeptide comprises an unnatural amino acid or D-amino acid.
 10. The pharmaceutical composition of claim 8, wherein the composition includes a sterile aqueous solution.
 11. The pharmaceutical composition of claim 8, wherein the composition is in a solvent-free form.
 12. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence of RRSLGHPEPLSNGRPQGNSRQWEQDEEEDEELTLKYGAK (SEQ ID NO:7) having 1-10 additional amino acids at either the N- and/or C-terminal end(s).
 13. The pharmaceutical composition of claim 1, wherein the polypeptide is at least 85% identical to RRSLGHPEPLSNGRPQGNSRQWEQDEEEDEELTLKYGAK (SEQ ID NO:7) having 1-5 conservative amino acid substitutions.
 14. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence of DEEEDELTLKYGAK (SEQ ID NO:17).
 15. The pharmaceutical composition of claim 14, wherein the polypeptide comprises an unnatural amino acid or D-amino acid.
 16. The pharmaceutical composition of claim 14, wherein the composition includes a sterile aqueous solution.
 17. The pharmaceutical composition of claim 14, wherein the composition is in a solvent-free form.
 18. The pharmaceutical composition of claim 1, wherein the polypeptide has the sequence of DEEEDELTLKYGAK (SEQ ID NO:17) having 1-10 additional amino acids at either the N- and/or C-terminal end(s).
 19. The pharmaceutical composition of claim 1, wherein the polypeptide is at least 85% identical to DEEEDELTLKYGAK (SEQ ID NO:17) having 1-2 conservative amino acid substitutions. 